Dual mode radiotracer and—therapeutics

ABSTRACT

The present invention relates to a ligand-SIFA-chelator conjugate, comprising, within in a single molecule three separate moieties: (a) one or more ligands which are capable of binding to a disease-relevant target molecule, (b) a silicon-fluoride acceptor (SIFA) moiety which comprises a covalent bond between a silicon atom and a fluorine atom, and (c) one or more chelating groups, optionally containing a chelated nonradioactive or radioactive cation.

RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 national stage filing ofInternational Application No. PCT/EP2018/070533, filed Jul. 30, 2018,which claims the benefit of priority to European Patent Application No.17183795.8, filed Jul. 28, 2017.

The present invention relates to a ligand-SIFA-chelator conjugate,comprising, within in a single molecule: (a) one or more ligands whichare capable of binding to a disease-relevant target molecule, (b) asilicon-fluoride acceptor (SIFA) moiety which comprises a covalent bondbetween a silicon and a fluorine atom and which can be labeled with ¹⁸Fby isotopic exchange of ¹⁹F by ¹⁸F or which is labeled with ¹⁸F, and (c)one or more chelating groups, optionally containing a chelatednonradioactive or radioactive cation.

In this specification, a number of documents including patentapplications and manufacturer's manuals are cited. The disclosure ofthese documents, while not considered relevant for the patentability ofthis invention, is herewith incorporated by reference in its entirety.More specifically, all referenced documents are incorporated byreference to the same extent as if each individual document wasspecifically and individually indicated to be incorporated by reference.

Prostate Cancer

Prostate Cancer (PCa) remained over the last decades the most commonmalignant disease in men with high incidence for poor survival rates.Due to its overexpression in prostate cancer (Silver et al., ClinicalCancer Research 3, 81-85 (1997)), prostate-specific membrane antigen(PSMA) or glutamate carboxypeptidase II (GCP II) proved its eligibilityas excellent target for the development of highly sensitiveradiolabelled agents for endoradiotherapy and imaging of PCa(Afshar-Oromieh et al., European journal of nuclear medicine andmolecular imaging 42, 197-209 (2015); Benešová et al., Journal ofNuclear Medicine 56, 914-920 (2015); Robu et al., Journal of NuclearMedicine, jnumed. 116.178939 (2016); Weineisen et al.; Journal ofNuclear Medicine 55, 1083-1083 (2014); Rowe et al., Prostate cancer andprostatic diseases (2016); Maurer et al., Nature Reviews Urology(2016)). Prostate-specific membrane antigen is an extracellularhydrolase whose catalytic center comprises two zinc(II) ions with abridging hydroxido ligand. It is highly upregulated in metastatic andhormone-refractory prostate carcinomas, but its physiologic expressionhas also been reported in kidneys, salivary glands, small intestine,brain and, to a low extent, also in healthy prostate tissue. In theintestine, PSMA facilitates absorption of folate by conversion ofpteroylpoly-γ-glutamate to pteroylglutamate (folate). In the brain, ithydrolyses N-acetyl-Laspartyl-L-glutamate (NAAG) to N-acetyl-L-aspartateand glutamate.

Prostate-specific membrane antigen (PSMA) is a type II transmembraneglycoprotein that is highly overexpressed on prostate cancer epithelialcells. Despite its name, PSMA is also expressed, to varying degrees, inthe neovasculature of a wide variety of nonprostate cancers. Among themost common nonprostate cancers to demonstrate PSMA expression includebreast, lung, colorectal, and renal cell carcinoma.

The general necessary structures of PSMA targeting molecules comprise abinding unit that encompasses a zinc-binding group (such as urea (Zhouet al., Nature Reviews Drug Discovery 4, 1015-1026 (2005)), phosphinateor phosphoramidate) connected to a P1′ glutamate moiety, which warrantshigh affinity and specificity to PSMA and is typically further connectedto an effector functionality (Machulkin et al., Journal of drugtargeting, 1-15 (2016)). The effector part is more flexible and to someextent tolerant towards structural modifications. The entrance tunnelaccommodates two other prominent structural features, which areimportant for ligand binding. The first one is an arginine patch, apositively charged area at the wall of the entrance funnel and themechanistic explanation for the preference of negatively chargedfunctionalities at the P1 position of PSMA. This appears to be thereason for the preferable incorporation of negative charged residueswithin the ligand-scaffold. An in-depth analysis about the effect ofpositive charges on PSMA ligands has been, to our knowledge, so far notconducted. Upon binding, the concerted repositioning of the arginineside chains can lead to the opening of an S1 hydrophobic accessorypocket, the second important structure that has been shown toaccommodate an iodo-benzyl group of several urea based inhibitors, thuscontributing to their high affinity for PSMA (Barinka et al., Journal ofmedicinal chemistry 51, 7737-7743 (2008)).

Zhang et al. discovered a remote binding site of PSMA, which can beemployed for bidentate binding mode (Zhang et al., Journal of theAmerican Chemical Society 132, 12711-12716 (2010)). The so calledarene-binding site is a simple structural motif shaped by the sidechains of Arg463, Arg511 and Trp541, and is part of the GCPII entrancelid. The engagement of the arene binding site by a distal inhibitormoiety can result in a substantial increase in the inhibitor affinityfor PSMA due to avidity effects. PSMA I&T was developed with theintention to interact this way with PSMA, albeit no crystal structureanalysis of binding mode is available. A necessary feature according toZhang et al. is a linker unit (Suberic acid in the case of PSMA I&T)which facilitates an open conformation of the entrance lid of GCPII andthereby enabling the accessibility of the arene-binding site. It wasfurther shown that the structural composition of the linker has asignificant impact on the tumor-targeting and biologic activity as wellas on imaging contrast and pharmacokinetics (Liu et al., Bioorganic &medicinal chemistry letters 21, 7013-7016 (2011)), properties which arecrucial for both high imaging quality and efficient targetedendoradiotherapy.

Two categories of PSMA targeting inhibitors are currently used inclinical settings. On the one side there are tracers with chelatingunits for radionuclide complexation such as PSMA I&T or relatedcompounds (Kiess et al., The quarterly journal of nuclear medicine andmolecular imaging 59, 241 (2015)). On the other side there are smallmolecules, comprising a targeting unit and effector molecules.

The most often used agents for selective PSMA imaging are PSMA HBED-CC(Eder et al., Bioconjugate chemistry 23, 688-697 (2012)), PSMA-617(Benešová et al., Journal of Nuclear Medicine 56, 914-920 (2015)) andPSMA I&T (Weineisen et al.; Journal of Nuclear Medicine 55, 1083-1083(2014)), which are predominantly labelled with ⁶⁸Ga (88.9% β⁺,E_(β+, max)=1.89 MeV, t_(1/2)=68 min). Among these ⁶⁸Ga-PSMA-HBED-CC(also known as ⁶⁸Ga-PSMA-11), is so far considered as the goldenstandard for PET imaging of PCa.

¹⁸F Labelling

Recently, several groups have focused on the development of novel¹⁸F-labelled urea-based inhibitors for PCa diagnosis. In contrast to theradiometal ⁶⁸Ga, which can be obtained from commercially distributed⁶⁸Ge/⁶⁸Ga radionuclide generators (⁶⁸Ge; t_(1/2)=270.8 d), theradioisotope ¹⁸F-fluoride (96.7% β⁺, E_(β+, max)=634 keV) requires anon-site cyclotron for its production. Despite this limitation, ¹⁸Foffers due to its longer half-live (t_(1/2)=109.8 min) and its lowerpositron energy, significant advantages in terms of routine-handling andimage quality. Additionally, there is the possibility for largescaleproduction in a cyclotron, which would be beneficial for a higherpatient throughput and reduction of production costs. The ¹⁸F-labelledurea-based PSMA inhibitor ¹⁸F-DCFPyl demonstrated promising results inthe detection of primary and metastatic PCa (Rowe et al., MolecularImaging and Biology, 1-9 (2016)) and superiority to ⁶⁸Ga-PSMA-HBED-CC ina comparative study (Dietlein et al., Molecular Imaging and Biology 17,575-584 (2015)). Based on the structure of PSMA-617, the ¹⁸F-labelledanalogue PSMA-1007 was recently developed, which showed comparabletumor-to-organ ratios (Cardinale et al., Journal of nuclear medicine:official publication, Society of Nuclear Medicine 58, 425-431 (2017);Giesel et al., European journal of nuclear medicine and molecularimaging 43, 1929-1930 (2016)). A comparative study with⁶⁸Ga-PSMA-HBED-CC revealed similar diagnostic accuracy of both tracersand a reduced urinary clearance of ¹⁸F-PSMA-1007, enabling a betterassessment of the prostate (Giesel et al., European journal of nuclearmedicine and molecular imaging 44, 678-688 (2017)).

An attractive approach for introducing ¹⁸F labels is the use of siliconfluoride acceptors (SIFA). Silicon fluoride acceptors are described, forexample, in Lindner et al., Bioconjugate Chemistry 25, 738-749 (2014).In order to preserve the silicon-fluoride bond, the use of siliconfluoride acceptors introduces the necessity of sterically demandinggroups around the silicone atom. This in turn renders silicon fluorideacceptors highly hydrophobic. In terms of binding to the targetmolecule, in particular to the target molecule which is PSMA, thehydrophobic moiety provided by the silicone fluoride acceptor may beexploited for the purpose of establishing interactions of theradio-diagnostic or -therapeutic compound with the hydrophobic pocketdescribed in Zhang et al., Journal of the American Chemical Society 132,12711-12716 (2010). Yet, prior to binding, the higher degree oflipophilicity introduced into the molecule poses a severe problem withrespect to the development of radiopharmaceuticals with suitable in vivobiodistribution, i.e. low unspecific binding in non-target tissue.

Failure to Solve the Hydrophobicity Problem

Despite many attempts, the hydrophobicity problem caused by siliconfluoride acceptors has not been satisfactorily solved in the prior art.

To explain further, Schirrmacher E. et al. (Bioconjugate Chem. 2007, 18,2085-2089) synthesized different ¹⁸F-labelled peptides using the highlyeffective labelling synthon p-(di-tert-butylfluorosilyl) benzaldehyde([¹⁸F]SIFA-A), which is one example of a silicon fluoride acceptor. TheSIFA technique resulted in an unexpectedly efficient isotopic ¹⁸F-¹⁸Fexchange and yielded the ¹⁸F-synthon in almost quantitative yields inhigh specific activities between 225 and 680 GBq/μmol (6081-18 378Ci/mmol) without applying HPLC purification. [¹⁸F]SIFA-benzaldehyde wasfinally used to label the N-terminal amino-oxy (N-AO) derivatizedpeptides AO-Tyr3-octreotate (AO-TATE), cyclo(fK(AO-N)RGD) andN-AO-PEG₂-[D-Tyr-Gln-Trp-Ala-Val-Ala-His-Thi-Nle-NH₂] (AO-BZH3, abombesin derivative) in high radiochemical yields. Nevertheless, thelabelled peptides are highly lipophilic (as can be taken from the HPLCretention times using the conditions described in this paper) and thusare unsuitable for further evaluation in animal models or humans.

In Wängler C. et al. (Bioconjugate Chem., 2009, 20 (2), pp 317-321), thefirst SIFA-based Kit-like radio-fluorination of a protein (rat serumalbumin, RSA) has been described. As a labelling agent,4-(di-tert-butyl[¹⁸F]fluorosilyl)benzenethiol (Si[¹⁸F]FA-SH) wasproduced by simple isotopic exchange in 40-60% radiochemical yield (RCY)and coupled the product directly to maleimide derivatized serum albuminin an overall RCY of 12% within 20-30 min. The technically simplelabelling procedure does not require any elaborated purificationprocedures and is a straightforward example of a successful applicationof Si-18F chemistry for in vivo imaging with PET. The time-activitycureves and μPET images of mice showed that most of the activity waslocalized in the liver, thus demonstrating that the labelling agent istoo lipophilic and directs the in vivo probe to hepatobiliary excretionand extensive hepatic metabolism.

Wängler C. et al. (see Bioconjug Chem. 2010 Dec. 15; 21(12):2289-96)subsequently tried to overcome the major drawback of the SIFAtechnology, the high lipophilicity of the resultingradiopharmaceuticals, by synthesizing and evaluating new SIFA-octreotateanalogues (SIFA-Tyr3-octreotate, SIFA-Asn(AcNH-β-Glc)-Tyr3-octreotateand SIFA-Asn(AcNH-β-Glc)-PEG-Tyr3-octreotate). In these compounds,hydrophilic linkers and pharmacokinetic modifiers were introducedbetween the peptide and the SIFA-moiety, i.e. a carbohydrate and a PEGlinker plus a carbohydrate. As a measure of lipophilicity of theconjugates, the log P(ow) was determined and found to be 0.96 forSIFA-Asn(AcNH-β-Glc)-PEG-Tyr³-octreotate and 1.23 forSIFA-Asn(AcNH-β-Glc)-Tyr³-octreotate. These results show that the highlipophilicity of the SIFA moiety can only be marginally compensated byapplying hydrophilic moieties. A first imaging study demonstratedexcessive hepatic clearance/liver uptake and thus has never beentransferred into a first human study.

Bernard-Gauthier et al. (Biomed Res Int. 2014; 2014:454503) reviews agreat plethora of different SIFA species that have been reported in theliterature ranging from small prosthetic groups and other compounds oflow molecular weight to labelled peptides and most recently affibodymolecules. Based on these data the problem of lipophilicity ofSIFA-based prosthetric groups has not been solved sofar; i.e. amethodology that reduces the overall lipophilicity of a SIFA conjugatedpeptide to a log D lower than approx −2.0 has not been described.

In Lindner S. et al. (Bioconjug Chem. 2014 Apr. 16; 25(4):738-49) it isdescribed that PEGylated bombesin (PESIN) derivatives as specific GRPreceptor ligands and RGD (one-letter codes for arginine-glycine-asparticacid) peptides as specific αvβ3 binders were synthesized and tagged witha silicon-fluorine-acceptor (SIFA) moiety. To compensate the highlipophilicity of the SIFA moiety various hydrophilic structuremodifications were introduced leading to reduced log D values.SIFA-Asn(AcNH-β-Glc)-PESIN, SIFA-Ser(β-Lac)-PESIN, SIFA-Cya-PESIN,SIFA-LysMe3-PESIN, SIFA-γ-carboxy-d-Glu-PESIN, SIFA-Cya2-PESIN,SIFA-LysMe3-γ-carboxy-d-Glu-PESIN, SIFA-(γ-carboxy-d-Glu)2-PESIN,SIFA-RGD, SIFA-γ-carboxy-d-Glu-RGD, SIFA-(γ-carboxy-d-Glu)2-RGD,SIFA-LysMe3-γ-carboxy-d-Glu-RGD. All of these peptides—already improvedand derivatized with the aim to reduce the lipophilicity—showed a log Dvalue in the range between +2 and −1.22.

In Niedermoser S. et al. (J Nucl Med. 2015 July; 56(7):1100-5), newlydeveloped ¹⁸F-SIFA- and ¹⁸F-SIFAlin-(SIFA=silicon-fluoride acceptor)modified TATE derivatives were compared with the current clinical goldstandard ⁶⁸Ga-DOTATATE for high-quality imaging of somatostatinreceptor-bearing tumors. For this purpose, ¹⁸F-SIFA-TATE and two quitecomplex analogues, ¹⁸F-SIFA-Glc-PEG1-TATE,¹⁸F-SIFAlin-Glc-Asp2-PEG1-TATE were developed. None of the agents showeda log D<−1.5.

In view of the above, the technical problem underlying the presentinvention can be seen in providing radio-diagnostics andradio-therapeutics which contain a silicone fluoride acceptor and whichare, at the same time, characterized by favourable in-vivo properties.

As will be become apparent in the following, the present inventionestablished a proof-of-principle using specific conjugates which bindwith high affinity to prostate-specific antigen (PSMA) as target.Accordingly, a further technical problem underlying the presentinvention can be seen in providing improved radio-therapeutics and-diagnostics for the medical indication which is cancer, preferablyprostate cancer.

These technical problems are solved by the subject-matter of the claims.Accordingly, in the first aspect, the present invention relates to aligand-SIFA-chelator conjugate, comprising, within in a single molecule:(a) one or more ligands which are capable of binding to adisease-relevant target molecule, (b) a silicon-fluoride acceptor (SIFA)moiety which comprises a covalent bond between a silicon and a fluorineatom and which can be labeled with ¹⁸F by isotopic exchange of ¹⁹F by¹⁸F or which is labeled with ¹⁸F, and (c) one or more chelating groups,optionally containing a chelated nonradioactive or radioactive cation.

The ligand-SIFA-chelator conjugate comprises three separate moieties.The three separate moieties are a) one or more ligands which are capableof binding to a disease-relevant target molecule, (b) a silicon-fluorideacceptor (SIFA) moiety which comprises a covalent bond between a siliconand a fluorine atom, and (c) one or more chelating groups, optionallycontaining a chelated nonradioactive or radioactive cation.

The fluorine atom on the SIFA moiety can be ¹⁹F or ¹⁸F.

Whilst certain ligands which are capable of binding to adisease-relevant target molecule may be cyclic peptides, such cyclicpeptides are not chelating groups as envisaged herein, as the problem ofthe hydrophobic SIFA moiety is not solved in the absence of a furtherchelating moiety. Thus compounds of the invention require a hydrophilicchelating group in addition to the ligands which are capable of bindingto a disease-relevant target molecule. The hydrophilic chelating groupis required to reduce the hydrophobic nature of the compounds caused bythe presence of the SIFA moiety.

The ligand in relation to the first aspect of the invention is definedin functional terms. This is the case because the present invention doesnot depend on the specific nature of the ligand in structural terms.Rather, a key aspect of the invention is the combination, within asingle molecule, of a silicon fluoride acceptor and a chelator or achelate. These two structural elements, SIFA and the chelator, exhibit aspatial proximity. Preferably, the shortest distance between two atomsof the two elements is less or equal 25 Å, more preferably less than 20Å and even more preferably less than 15 Å. Alternatively or in addition,it is preferred that not more than 25 covalent bonds separate an atom ofthe SIFA moiety and an atom the chelator, preferably not more than 20chemical bonds and even more preferably not more than 15 chemical bonds.

The cation in accordance with item (c) of the first aspect is aradioactive or non-radioactive cation. It is preferably a radioactive ornon-radioactive metal cation, and more preferably a radioactive metalcation. Examples are given further below.

As a consequence, conjugates fall under the terms of the first aspectwhich are radioactively labelled at both the SIFA moiety and thechelating group, molecules which are radioactive labelled at only one ofthe two sides, as well as molecules which are not radiolabelled at all.In the latter case, the chelating group may be either a complex of acold (non-radioactive) ion or may devoid of any ion.

The present inventors surprisingly discovered that placement of thesilicone fluoride acceptor in the neighbourhood of a hydrophilicchelator such as, but not limited to, DOTAGA or DOTA, shields orcompensates efficiently the lipophilicity of the SIFA moiety to anextent which shifts the overall hydrophobicity of the radio-therapeuticor -diagnostic compound in a range which renders the compound suitablefor in-vivo administration.

In addition, the combination of the use of a chelator and an isotopicexchange on SIFA by means of ¹⁸F-fluoride also results in “paired”diagnostic tracers that can either be used as [¹⁸F][^(nat)Ion]tracers atcenters with onsite cyclotron or centers that obtain ¹⁸F-fluoride byshipment from cyclotron centers, whereas in centers, that do not haveaccess to ¹⁸F-fluoride but have access to radioisotope generators, suchas a Ge-68/Ga-68 generator, the corresponding versions, e.g.[^(nat)F][⁶⁸Ga]tracers can be used.

Importantly, in both cases, the chemically identical radiopharmaceuticalis injected, and thus no differences in the in vivo behavior areexpected. Whereas currently, due to chemical differences, the clinicaldata of a ¹⁸F-labelled compound provided by a patient cohort at one sitecannot be directly compared with the clinical data of a ⁶⁸Ga-analogueprovided by another group at another site, radiopharmaceuticals and/ordiagnostics according to the invention can be directly compared and thuswill allow to link such data (e.g. data from a center in Europe workingwith F-18 and another center in India working with Ga-68).

Furthermore, when suitably selected, the chelate can also be used forlabelling with a therapeutic isotope, such as the beta-emitting isotopesLu-177, Y-90, or the alpha emitting isotope Ac-225, thus allowing toexpand the concept of “paired” tracers to bridge diagnostic([¹⁸F][^(nat)Lu]tracers) and therapeutic radiopharmaceuticals([^(nat)F][¹⁷⁷Lu].

A further advantage of the compounds, especially of PSMA targetedcompounds of the present invention is their surprisingly lowaccumulation in the kidneys of mice when compared to other PSMA targetedradiopharmaceuticals, such as PSMA I&T. Without wishing to be bound by aparticular theory, it seems to be the combination of the structuralelement SIFA with a chelator which provides for the unexpected reductionof accumulation in the kidneys.

In terms of lipophilicity/hydrophilicity, the log P value (sometimesalso referred to as log D value) is an art-established measure.

The term “lipophilicity” relates to the strength of being dissolved in,or be absorbed in lipid solutions, or being adsorbed at a lipid-likesurface or matrix. It denotes a preference for lipids (literal meaning)or for organic or apolar liquids or for liquids, solutions or surfaceswith a small dipole moment as compared to water. The term“hydrophobicity” is used with equivalent meaning herein. The adjectiveslipophilic and hydrophobic are used with corresponding meaning to thesubstantives described above.

The mass flux of a molecule at the interface of two immiscible orsubstantially immiscible solvents is governed by its lipophilicity. Themore lipophilic a molecule is, the more soluble it is in the lipophilicorganic phase. The partition coefficient of a molecule that is observedbetween water and n-octanol has been adopted as the standard measure oflipophilicity. The partition coefficient P of a species A is defined asthe ratio P=[A]_(n-octanol)/[A]_(water). A figure commonly reported isthe log P value, which is the logarithm of the partition coefficient. Incase a molecule is ionizable, a plurality of distinct microspecies(ionized and not ionized forms of the molecule) will in principle bepresent in both phases. The quantity describing the overalllipophilicity of an ionizable species is the distribution coefficient D,defined as the ratio D=[sum of the concentrations of allmicrospecies]_(n-octanol)/[sum of the concentrations of allmicrospecies]_(water). Analogous to log P, frequently the logarithm ofthe distribution coefficient, log D, is reported. Often, a buffersystem, such as phosphate buffered saline is used as alternative towater in the above described determination of log P.

If the lipophilic character of a substituent on a first molecule is tobe assessed and/or to be determined quantitatively, one may assess asecond molecule corresponding to that substituent, wherein said secondmolecule is obtained, for example, by breaking the bond connecting saidsubstituent to the remainder of the first molecule and connecting (the)free valence(s) obtained thereby to hydrogen(s).

Alternatively, the contribution of the substituent to the log P of amolecule may be determined. The contribution π_(Xx) of a substituent Xto the log P of a molecule R-X is defined as π_(Xx)=log P_(R-X)−logP_(R-H), wherein R—H is the unsubstituted parent compound.

Values of P and D greater than one as well as log P, log D and π×xvalues greater than zero indicate lipophilic/hydrophobic character,whereas values of P and D smaller than one as well as log P, log D andπ×x values smaller than zero indicate hydrophilic character of therespective molecules or substituents.

The above described parameters characterizing the lipophilicity of thelipophilic group or the entire molecule according to the invention canbe determined by experimental means and/or predicted by computationalmethods known in the art (see for example Sangster, Octanol-waterPartition Coefficients: fundamentals and physical chemistry, John Wiley& Sons, Chichester. (1997)).

In a preferred embodiment, the log P value of the compounds of theinvention is between −5 and −1.5. It is particularly preferred that thelog P value is between −3.5 and −2.0.

In a preferred embodiment, a ligand in accordance with the inventioncomprises or consists of a peptide, a peptidomimetic or a substitutedurea, substituents including amino acids. It is understood that a ligandwhich comprises a peptide or peptidomimetic also comprises anon-peptidic and non-peptidomimetic part. In terms of molecular weight,preference is given to molecular weights below 15 kDa, below 10 kDa orbelow 5 kDa. Accordingly, small proteins are also embraced by the term“ligand”. Target molecules are not particularly limited and includeenzymes, receptors, epitopes, transporters, cell surface molecules andproteins of the extracellular matrix. Preferred are targets which aredisease relevant. Particularly preferred are targets which are causallyinvolved in a given disease, or which are highly overexpressed in agiven disease and/or the inhibition of which can cause a beneficialeffect in a patient suffering from a given disease. The ligands arepreferably high affinity ligands with preferable affinity, expressed asIC₅₀, being below 50 nM, below 20 nM or below 5 nM.

Especially preferred are those ligands which bind with high affinity todisease-relevant target molecules or disease-relevant biomoleculesincluding, but not limited to somatostatin receptors, bombesinreceptors, chemokine receptors, integrin receptors, cholecystokininreceptors, melanocortin receptors, vasoactive intestinal peptidereceptors, neurotensin receptors, neuropeptide Y receptors, neurokininreceptors, glucacon-like peptide 1 receptors, Her2− receptors, PD-L1,PD-1, gonadotropin releasing hormone receptors and prostate-specificmembrane antigen (PSMA).

The term “disease-relevant” refers preferably to a causal involvement ina disease.

Preferably, the silicon-fluoride acceptor (SIFA) moiety has thestructure represented by formula (I):

wherein R^(1S) and R^(2S) are independently a linear or branched C3 toC10 alkyl group, preferably R^(1S) and R^(2S) are selected fromisopropyl and tert-butyl, and are more preferably R^(1S) and R^(2S) aretert-butyl; R^(3S) is a C1 to C20 hydrocarbon group which may compriseone or more aromatic and one or more aliphatic units and/or up to 3heteroatoms selected from O and S, preferably R^(3S) is a C6 to C10hydrocarbon group which comprises an aromatic ring and which maycomprise one or more aliphatic units; more preferably R^(3S) is a phenylring, and most preferably, R^(3S) is a phenyl ring wherein theSi-containing substituent and the bond marked by

are in a para-position, and wherein the SIFA moiety is attached to theremainder of the conjugate via the bond marked by

.

More preferably, the silicon-fluoride acceptor (SIFA) moiety has thestructure represented by formula (Ia):

wherein t-Bu indicates a tert-butyl group.

A preferred chelating group comprises at least one of the following (i),(ii) or (iii).

(i) A macrocyclic ring structure with 8 to 20 ring atoms of which 2 ormore, more preferably 3 or more, are selected from oxygen atoms ornitrogen atoms. Preferably, 6 or less ring atoms are selected fromoxygen atoms or nitrogen atoms. Especially preferred is that 3 or 4 ringatoms are nitrogen atoms or oxygen atoms. Among the oxygen and nitrogenatoms, preference is given to the nitrogen atoms. In combination withthe macrocyclic ring structure, the preferred chelating group maycomprise 2 or more, such as 2 to 6, preferably 2 to 4, carboxyl groupsand/or hydroxyl groups. Among the carboxyl groups and the hydroxylgroups, preference is given to the carboxyl groups.

(ii) An acyclic, open chain chelating structure with 8 to 20 main chain(back bone) atoms of which 2 or more, more preferably 3 or more areheteroatoms selected from oxygen atoms or nitrogen atoms. Preferably, 6or less back bone atoms are selected from oxygen atoms or nitrogenatoms. Among the oxygen and nitrogen atoms, preference is given to thenitrogen atoms. More preferably, the open chain chelating structure is astructure which comprises a combination of 2 or more, more preferably 3or more heteroatoms selected from oxygen atoms or nitrogen atoms, and 2or more, such as 2 to 6, preferably 2 to 4, carboxyl groups and/orhydroxyl groups. Among the carboxyl groups and the hydroxyl groups,preference is given to the carboxyl groups.

(iii) A branched chelating structure containing a quarternary carbonatom. Preferably the quarternary carbon atom is substituted with 3identical chelating groups in addition to the SIFA/ligand moiety. Thesubstituted chelating groups can comprise an amide. The substitutedchelating groups can comprise an aromatic group. The substitutedchelating groups can comprise a hydroxypyridinone.

In preferred specific examples, the chelating group is a residue of achelating agent selected frombis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CBTE2a),cyclohexyl-1,2-diaminetetraacetic acid (CDTA),4-(1,4,8,11-tetraazacyclotetradec-1-yl)-methylbenzoic acid (CPTA),N′-[5-[acetyl(hydroxy)amino]pentyl]-N-[5-[[4-[5-aminopentyl-(hydroxy)amino]-4-oxobutanoyl]amino]pentyl]-N-hydroxybutandiamide(DFO), 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecan(DO2A) 1,4,7,10-tetracyclododecan-N,N′,N″,N″′-tetraacetic acid (DOTA),α-(2-carboxyethyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid (DOTAGA), 1,4,7,10 tetraazacyclododecane N, N′,N″, N″′1,4,7,10-tetra(methylene) phosphonic acid (DOTMP),N,N′-dipyridoxylethylendiamine-N,N′-diacetate-5,5′-bis(phosphat) (DPDP),diethylene triamine N,N′,N″ penta(methylene) phosphonic acid (DTMP),diethylenetriaminepentaacetic acid (DTPA),ethylenediamine-N,N′-tetraacetic acid (EDTA),ethyleneglykol-O,O-bis(2-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA),N,N-bis(hydroxybenzyl)-ethylenediamine-N,N′-diacetic acid (HBED),hydroxyethyldiaminetriacetic acid (HEDTA),1-(p-nitrobenzyl)-1,4,7,10-tetraazacyclodecan-4,7,10-triacetate(HP-DOA3), 6-hydrazinyl-N-methylpyridine-3-carboxamide (HYNIC), tetra3-hydroxy-N-methyl-2-pyridinone chelators(4-((4-(3-(bis(2-(3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamido)ethyl)amino)-2-((bis(2-(3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamido)ethyl)amino)methyl)propyl)phenyl)amino)-4-oxobutanoicacid), abbreviated as Me-3,2-HOPO, 1,4,7-triazacyclononan-1-succinicacid-4,7-diacetic acid (NODASA),1-(1-carboxy-3-carboxypropyl)-4,7-(carbooxy)-1,4,7-triazacyclononane(NODAGA), 1,4,7-triazacyclononanetriacetic acid (NOTA),4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane(TE2A), 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA),tris(hydroxypyridinone) (THP), terpyridin-bis(methyleneamintetraaceticacid (TMT),1,4,7-triazacyclononane-1,4,7-tris[methylene(2-carboxyethyl)phosphinicacid] (TRAP), 1,4,7,10-tetraazacyclotridecan-N,N′,N″,N″′-tetraaceticacid (TRITA),3-[[4,7-bis[[2-carboxyethyl(hydroxy)phosphoryl]methyl]-1,4,7-triazonan-1-yl]methyl-hydroxy-phosphoryl]propanoicacid, and triethylenetetraaminehexaacetic acid (TTHA), which residue isprovided by covalently binding a carboxyl group contained in thechelating agent to the remainder of the conjugate via an ester or anamide bond.

Particular chelators are shown below:

Among the above exemplary chelating agents, particular preference isgiven to a chelating agent selected from TRAP, DOTA and DOTAGA.

Metal- or cation-chelating macrocyclic and acyclic compounds arewell-known in the art and available from a number of manufacturers.While the chelating moiety in accordance with the present invention isnot particularly limited, it is understood that numerous moieties can beused in an off-the-shelf manner by a skilled person without further ado.

The chelating group may comprise a chelated cation which may beradioactive or non-radioactive, preferably a chelated metal cation whichmay be radioactive or non-radioactive.

More preferred is a chelated radioactive metal isotope.

Preferred examples of cations that may be chelated by the chelatinggroup are the cations of ⁴³Sc, ⁴⁴Sc, ⁴⁷Sc, ⁵¹Cr, ^(52m)Mn, ⁵⁸Co, ⁵²Fe,⁵⁶Ni, ⁵⁷Ni, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁶Ga, ⁶⁷Ga ⁶⁸Ga, ⁸⁹Zr, ⁹⁰Y, ⁸⁹Y, <Tc,^(99m)Tc, ⁹⁷Ru, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹¹¹Ag, ^(110m)In, ¹¹¹In, ^(113m)In,^(114m)In, ^(117m)Sn, ¹²¹Sn, ¹²⁷Te, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵¹Pm, ¹⁴⁹Tb,¹⁵²Tb, ¹⁵⁵Tb, ¹⁶¹Tb, ¹⁵³Sm, ¹⁵⁷Gd, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁶⁵Dy, ¹⁶⁹Er, ¹⁶⁹Yb,¹⁷⁵Yb, ¹⁷²Tm, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹¹Pt, ¹⁹⁷Hg, ¹⁹⁸Au, ¹⁹⁹Au, ²¹²Pb,²⁰³Pb, ²¹¹At., ²¹²Bi, ²¹³Bi, ²²³Ra, ²²⁵Ac, ²²⁷Th, a cationic moleculecomprising ¹⁸F or a cation such as ¹⁸F-[AIF]²⁺; more preferably thecations of ⁴⁴Sc, ⁴⁷Sc, ⁶⁴Cu, ⁶⁷Cu, ⁶⁸Ga, ⁹⁰Y, 111In, ¹⁶¹Tb, ¹⁶⁶Ho,¹⁷⁷Lu, ¹⁸⁸Re, ²¹²Pb, ²¹²Bi, ²¹³Bi, ²²⁵Ac, and ²²⁷Th or a cationicmolecule comprising ¹⁸F.

Using PSMA binders as an example, the present inventors reduced theabove disclosed invention to practice. This is the subject-matter of thepreferred aspects and embodiments disclosed in the following. Yet, thesurprising finding made by the present inventors—compensation of thelipophilicity of the SIFA moiety to a surprising extent—is not limitedto molecules comprising a PSMA binder.

Accordingly, the ligand is preferably capable of binding toprostate-specific membrane antigen (PSMA).

More preferably, the ligand has the structure represented by formula(II):

wherein m is an integer of 2 to 6, preferably 2 to 4, more preferably 2;n is an integer of 2 to 6, preferably 2 to 4, more preferably 2 or 3;R^(1L) is CH₂, NH or O, preferably NH; R^(3L) is CH₂, NH or O,preferably NH; R^(2L) is C or P(OH), preferably C; and wherein theligand is attached to the remainder of the conjugate via the bond markedby

.

The ligand can have the structure represented by formula (IIa):

wherein n is an integer of 2 to 6; and wherein the ligand is attached tothe remainder of the conjugate via the bond marked by

.

A number of PSMA binders are known in the art which are all suitable inaccordance with the invention. The above preferred embodiment is astructural definition of a preferred group of PSMA binders.

It is particularly preferred that the conjugate of the first aspect is acompound of formula (III):

or a pharmaceutically acceptable salt thereof, wherein:SIFA is a silicon-fluoride acceptor (SIFA) moiety which comprises acovalent bond between a silicon and a fluorine atom and which can belabeled with ¹⁸F by isotopic exchange of ¹⁹F by ¹⁸F or which is labeledwith ¹⁸F; preferably SIFA is the SIFA moiety of formula (I) and morepreferably of formula (Ia) defined above;m is an integer of 2 to 6, preferably 2 or 3, more preferably 2;n is an integer of 2 to 6, preferably 2 or 3, more preferably 2 or 4;R^(1L) is CH₂, NH or O, preferably NH;R^(3L) is CH₂, NH or O, preferably NH;R^(2L) is C or P(OH), preferably C;X¹ is selected from an amide bond, an ether bond, a thioether bond, anester bond, a thioester bond, an urea bridge, and an amine bond,preferably an amide bond;X² is selected from an amide bond, an ether bond, a thioether bond, anester bond, a thioester bond, an urea bridge, and an amine bond,preferably an amide bond;L¹ is a divalent linking group with a structure selected from anoligoamide, an oligoether, an oligothioether, an oligoester, anoligothioester, an oligourea, an oligo(ether-amide), anoligo(thioether-amide), an oligo(ester-amide), anoligo(thioester-amide), oligo(urea-amide), an oligo(ether-thioether), anoligo(ether-ester), an oligo(ether-thioester), an oligo ether-urea), anoligo(thioether-ester), an oligo(thioether-thioester), anoligo(thioether-urea), an oligo(ester-thioester), an oligo(ester-urea),and an oligo(thioester-urea), preferably with a structure selected froman oligoamide and an oligo(ester-amide).L¹ can be optionally substituted with one or more substitutentsindependently selected from —OH, —OCH₃, —COOH, —COOCH₃, —NH₂, and—NHC(NH)NH₂.X³ is selected from an amide bond, and ester bond, an ether, an amine,and a linking group of the formula:

wherein the bond marked by

at the NH group is bound to R^(B) and the other bond marked by

is bound to SIFA; preferably X³ is an amide bond; R^(B) is a trivalentcoupling group;X⁴ is selected from an amide bond, an ether bond, a thioether bond, andester bond, a thioester bond, a urea bridge, an amine bond, a linkinggroup of the formula:

wherein the amide bond marked by

is formed with the chelating group, and the other bond marked by

bound to R^(B); and a linking group of the formula:

wherein the bond marked by

at the carbonyl end is formed with the chelating group, and the otherbond marked by

is bound to R^(B); preferably X⁴ is an amide bond; R^(CH) is chelatinggroup optionally containing a chelated radioactive or nonradioactivecation, preferably a radioactive or nonradioactive metal cation, whereinpreferred embodiments of said chelating group and of the optionalchelated cation are as defined above.

The term “oligo” as used in oligoamide, oligoether, oligothioether,oligoester, oligothioester, oligourea, oligo(ether-amide),oligo(thioether-amide), oligo(ester-amide), oligo(thioester-amide),oligo(urea-amide), oligo(ether-thioether), oligo(ether-ester),oligo(ether-thioester), oligo (ether-urea), oligo(thioether-ester),oligo(thioether-thioester), oligo(thioether-urea),oligo(ester-thioester), oligo(ester-urea), and oligo(thioester-urea) ispreferably to be understood as referring to a group wherein 2 to 20,more preferably wherein 2 to 10 subunits are linked by the type of bondsspecified in the same terms. As will be understood by the skilledreader, where two different types of bonds are indicated in brackets,both types of bonds are contained in the concerned group (e.g. in “oligo(ester-amide)”, ester bonds and amide bonds are contained).

It is preferred that L¹ comprises a total of 1 to 5, more preferably atotal of 1 to 3, and most preferably a total of 1 or 2 amide and/orester bonds, preferably amide bonds, within its backbone.

The term oligoamide therefore describes a moiety having a chain of CH₂or CHR groups interspersed with groups selected from NHCO or CONH. Eachoccurrence of the R moiety is an optional substituent selected from —OH,—OCH₃, —COOH, —COOCH₃, —NH₂, and —NHC(NH)NH₂.

It is also preferred that —X¹-L¹-X²— represents one of the followingstructures (L-1) and (L-2):—NH—C(O)—R⁶—C(O)—NH—R⁷—NH—C(O)—  (L-1)—C(O)—NH—R⁸—NH—C(O)—R⁹—C(O)—NH—R¹⁰—NH—C(O)—  (L-2)wherein R⁶ to R¹⁰ are independently selected from C2 to C10 alkylene,preferably linear C2 to C10 alkylene, which alkylene groups may each besubstituted by one or more substitutents independently selected from—OH, —OCH₃, —COOH, —COOCH₃, —NH₂, and —NHC(NH)NH₂.

Especially preferred is that the total number of carbon atoms in R⁶ andR⁷ is 4 to 20, more preferably 4 to 16, without carbon atoms containedin optional substituents. Especially preferred is that the total numberof carbon atoms in R⁸ to R¹⁰, is 6 to 20, more preferably 6 to 16,without carbon atoms contained in optional substituents.

It is particularly preferred that —X¹-L¹-X²— represents one of thefollowing structures (L-3) and (L-4):—NH—C(O)—R¹¹—C(O)—NH—R¹²—CH(COOH)—NH—C(O)—  (L-3)—C(O)—NH—CH(COOH)—R¹³—NH—C(O)—R¹⁴—C(O)—NH—R¹⁵—CH(COOH)—NH—C(O)—  (L-4)wherein R¹¹ to R¹⁵ are independently selected from C2 to C8 alkylene,preferably linear C2 to C8 alkylene.

Especially preferred is that the total number of carbon atoms in R¹¹ andR¹² or R¹³ to R¹⁵, respectively, is 8 to 18, more preferably 8 to 12,yet more preferably 9 or 10.

Preferably, R^(B) has the structure represented by formula (IV):

wherein: A is selected from N, CR¹⁶, wherein R¹⁶ is H or C1-C6 alkyl,and a 5 to 7 membered carbocyclic or heterocyclic group; preferably A isselected from N and CH, and more preferably A is CH; the bond marked by

at (CH₂)_(a) is formed with X², and a is an integer of 0 to 4,preferably 0 or 1, and most preferably 0; the bond marked by

at (CH₂)_(b) is formed with X³, and b is an integer of 0 to 4,preferably of 0 to 2, and more preferably 0 or 1; and the bond marked by

at (CH₂)_(c) is formed with X⁴, and c is an integer of 0 to 4,preferably of 0 to 2, and more preferably 0 or 1.

Even more preferred as a conjugate in accordance with the invention is acompound of formula (IIIa):

or a pharmaceutically acceptable salt thereof, wherein m, n, R^(1L),R^(2L), R^(3L), X¹, L¹, b, c, X⁴ and R^(CH) are as defined above,including all preferred embodiments thereof.

It is preferred for the compound of formula (IIIa) that b+c≥1.

It is also preferred for the compound of formula (IIIa) that b+c≤3.

It is more preferred for the compound of formula (IIIa) that b is 1 andc is 0.

It is also preferred for the compound of formula (III) that —X⁴—R^(CH)represents a residue of a chelating agent selected from DOTA and DOTAGAbound with one of its carboxylic groups via an amide bond to theremainder of the conjugate.

In a preferred embodiment of the compound of formula (III), saidcompound is a compound of formula (IIIb):

or a pharmaceutically acceptable salt thereof, wherein m, n, R^(1L),R^(2L), R^(3L), X¹, L¹, b, c, X⁴ and R^(CH) are as defined above; and ris 0 or 1.

Especially preferred is that —N(H)—R^(CH) represents a residue of achelating agent selected from DOTA and DOTAGA bound with one of itscarboxylic groups via an amide bond to the remainder of the conjugate.

Most preferred compounds of the invention are the following:

and isomers thereof:

and isomers thereof

and isomers thereof

and isomers thereof

and isomers thereof:

and isomers thereof:

Preferred labelling schemes for these most preferred compounds are asdefined herein above.

In a further aspect, the present invention provides a pharmaceuticalcomposition comprising or consisting of one or more conjugates orcompounds of the invention as disclosed herein above.

In a further aspect, the present invention provides a diagnosticcomposition comprising or consisting of one or more conjugates orcompounds of the invention as disclosed herein above.

In a further aspect, the present invention provides a therapeuticcomposition comprising or consisting of one or more conjugates orcompounds of the invention as disclosed herein above.

The pharmaceutical composition may further comprise pharmaceuticallyacceptable carriers, excipients and/or diluents. Examples of suitablepharmaceutical carriers, excipients and/or diluents are well known inthe art and include phosphate buffered saline solutions, water,emulsions, such as oil/water emulsions, various types of wetting agents,sterile solutions etc. Compositions comprising such carriers can beformulated by well known conventional methods. These pharmaceuticalcompositions can be administered to the subject at a suitable dose.Administration of the suitable compositions may be effected by differentways, e.g., by intravenous, intraperitoneal, subcutaneous,intramuscular, topical, intradermal, intranasal or intrabronchialadministration. It is particularly preferred that said administration iscarried out by injection and/or delivery, e.g., to a site in thepancreas or into a brain artery or directly into brain tissue. Thecompositions may also be administered directly to the target site, e.g.,by biolistic delivery to an external or internal target site, like thepancreas or brain. The dosage regimen will be determined by theattending physician and clinical factors. As is well known in themedical arts, dosages for any one patient depends upon many factors,including the patient's size, body surface area, age, the particularcompound to be administered, sex, time and route of administration,general health, and other drugs being administered concurrently.Pharmaceutically active matter may be present in amounts between 0.1 ngand 10 mg/kg body weight per dose; however, doses below or above thisexemplary range are envisioned, especially considering theaforementioned factors.

In a further aspect, the present invention provides one or morecompounds of the invention as disclosed herein above for use inmedicine.

Preferred uses in medicine are in nuclear medicine such as nucleardiagnostic imaging, also named nuclear molecular imaging, and/ortargeted radiotherapy of diseases associated with an overexpression,preferably of PSMA on the diseased tissue.

In a further aspect, the present invention provides a compound of theinvention as defined herein above for use in a method of diagnosingand/or staging cancer, preferably prostate cancer. Prostate cancer isnot the only cancer to express PSMA. Nonprostate cancers to demonstratePSMA expression include breast, lung, colorectal, and renal cellcarcinoma.

Thus any compound described herein having a PSMA binding moiety can beused in the diagnosis, imaging or treatment of a cancer having PSMAexpression.

Preferred indications are the detection or staging of cancer, such as,but not limited high grade gliomas, lung cancer and especially prostatecancer and metastasized prostate cancer, the detection of metastaticdisease in patients with primary prostate cancer of intermediate-risk tohigh-risk, and the detection of metastatic sites, even at low serum PSAvalues in patients with biochemically recurrent prostate cancer. Anotherpreferred indication is the imaging and visualization of neoangiogensis.

In terms of medical indications to be subjected to therapy, especiallyradiotherapy, cancer is a preferred indication. Prostate cancer is aparticularly preferred indication.

In a further aspect, the present invention provides a conjugate orcompound of the invention as defined herein above for use in a method ofdiagnosing and/or staging cancer, preferably prostate cancer.

The present disclosure furthermore relates to the following items.

-   1. A ligand-SIFA-chelator conjugate, comprising, within in a single    molecule:    -   (a) one or more ligands which are capable of binding to a        disease-relevant target molecule,    -   (b) a silicon-fluoride acceptor (SIFA) moiety which comprises a        covalent bond between a silicon and a fluorine atom and which        can be labeled with ¹⁸F by isotopic exchange of ¹⁹F by ¹⁸F or        which is labeled with ¹⁸F, and    -   (c) one or more chelating groups, optionally containing a        chelated nonradioactive or radioactive cation.-   2. The conjugate in accordance with item 1, wherein the    silicon-fluoride acceptor (SIFA) moiety has the structure    represented by formula (I):

wherein

-   -   R^(1S) and R^(2S) are independently a linear or branched C3 to        C10 alkyl group, preferably    -   R^(1S) and R^(2S) are selected from isopropyl and tert-butyl,        and are more preferably R^(1S) and R^(2S) are tert-butyl;    -   R^(3S) is a C1 to C20 hydrocarbon group which may comprise one        or more aromatic and one or more aliphatic units and/or up to 3        heteroatoms selected from O and S, preferably R^(3S) is a C6 to        C10 hydrocarbon group which comprises an aromatic ring and which        may comprise one or more aliphatic units; more preferably R^(3S)        is a phenyl ring, and most preferably, R^(3S) is a phenyl ring        wherein the Si-containing substituent and the bond marked by        in a para-position, and wherein the SIFA moiety is attached to        the remainder of the conjugate via the bond marked by        .

-   3. The conjugate in accordance with item 2, wherein the    silicon-fluoride acceptor (SIFA) moiety has the structure    represented by formula (Ia):

wherein t-Bu indicates a tert-butyl group.

-   4. The conjugate in accordance with any of items 1 to 3, wherein the    chelating group comprises at least one of    -   (i) a macrocyclic ring structure with 8 to 20 ring atoms of        which 2 or more, preferably 3 or more, are selected from oxygen        atoms and nitrogen atoms; and    -   (ii) an acyclic, open chain chelating structure with 8 to 20        main chain atoms of which 2 or more, preferably 3 or more are        heteroatoms selected from oxygen atoms and nitrogen atoms.-   5. The conjugate in accordance with any of items 1 to 3, wherein the    chelating group is a residue of a chelating agent selected from    bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane    (CBTE2a), cyclohexyl-1,2-diaminetetraacetic acid (CDTA),    4-(1,4,8,11-tetraazacyclotetradec-1-yl)-methylbenzoic acid (CPTA),    N′-[5-[acetyl(hydroxy)amino]pentyl]-N-[5-[[4-[5-aminopentyl-(hydroxy)amino]-4-oxobutanoyl]amino]pentyl]-N-hydroxybutandiamide    (DFO),    4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecan    (DO2A) 1,4,7,10-tetracyclododecan-N,N′,N″,N″′-tetraacetic acid    (DOTA),    N,N′-dipyridoxylethylendiamine-N,N′-diacetate-5,5′-bis(phosphat)    (DPDP), diethylenetriaminepentaacetic acid (DTPA),    ethylenediamine-N,N′-tetraacetic acid (EDTA),    ethyleneglykol-O,O-bis(2-aminoethyl)-N,N,N′,N′-tetraacetic acid    (EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N′-diacetic acid    (HBED), hydroxyethyldiaminetriacetic acid (HEDTA),    1-(p-nitrobenzyl)-1,4,7,10-tetraazacyclodecan-4,7,10-triacetate    (HP-DOA3), 6-hydrazinyl-N-methylpyridine-3-carboxamide (HYNIC),    1,4,7-triazacyclononan-1-succinic acid-4,7-diacetic acid (NODASA),    1-(1-carboxy-3-carboxypropyl)-4,7-(carbooxy)-1,4,7-triazacyclononane    (NODAGA), 1,4,7-triazacyclononanetriacetic acid (NOTA),    4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane    (TE2A), 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid    (TETA), terpyridin-bis(methyleneamintetraacetic acid (TMT),    1,4,7,10-tetraazacyclotridecan-N,N′,N″,N″′-tetraacetic acid (TRITA),    and triethylenetetraaminehexaacetic acid (TTHA); which residue is    provided by covalently binding a carboxyl group contained in the    chelating agent to the remainder of the conjugate via an ester or an    amide bond, preferably via an amide bond.-   6. The conjugate in accordance with item 5, wherein the chelating    agent is selected from DOTA and DOTAGA.-   7. The conjugate in accordance with any of items 1 to 6, wherein the    chelating group comprises a chelated cation, preferably a chelated    radioactive cation selected from ⁴⁴Sc, ⁴⁷Sc, ⁵¹Cr, ^(52m)Mn, 58Co,    ⁵²Fe, ⁵⁶Ni, ⁵⁷Ni, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁶Ga, ⁶⁸Ga, ⁶⁷Ga, ⁸⁹Zr, ⁹⁰Y,    ⁸⁹Y, <Tc, ^(99m)TC, ⁹⁷Ru, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹¹¹Ag, ^(110m)In, ¹¹¹In,    ^(113m)In, ^(114m)In, ^(117m)Sn, ¹²¹Sn, ¹²⁷Te, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm,    ¹⁵¹Pm, ¹⁴⁹Tb, ¹⁵³Sm, ¹⁵⁷Gd, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁶⁵Dy, ¹⁶⁹Er, ¹⁶⁹Yb,    ¹⁷⁵Yb, ¹⁷²Tm, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹¹Pt, ¹⁹⁷Hg, ¹⁹⁸Au, ¹⁹⁹Au,    ²¹²Pb, ²⁰³Pb, ²¹¹At., ²¹²Bi, ²¹³Bi, ²²³Ra, ²²⁵Ac, ²²⁷Th, a cationic    molecule comprising ¹⁸F or a cation such as ¹⁸F-[AIF]²⁺; more    preferably the cations of ⁴⁴Sc, ⁴⁷Sc, ⁶⁴Cu, ⁶⁷Cu, ⁶⁸Ga, ⁹⁰Y, ¹¹¹In,    ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁸Re, ²¹²Pb, ²¹²Bi, ²¹³Bi, ²²⁵Ac, and ²²⁷Th or    a cationic molecule comprising ¹⁸F.-   8. The conjugate in accordance with any of items 1 to 8, wherein the    ligand is capable of binding to PSMA.-   9. The conjugate in accordance with any of items 1 to 8, wherein the    ligand has the structure represented by formula (II):

-   -   wherein:    -   m is an integer of 2 to 6, preferably 2 to 4, more preferably 2;    -   n is an integer of 2 to 6, preferably 2 to 4, more preferably 2        or 3;    -   R^(1L) is CH₂, NH or O, preferably NH;    -   R^(3L) is CH₂, NH or O, preferably NH;    -   R^(2L) is C or P(OH), preferably C;    -   and wherein the ligand is attached to the remainder of the        conjugate via the bond marked by        .

-   10. The conjugate in accordance with any of items 1 to 9, which is a    compound of formula (III):

-   -   or a pharmaceutically acceptable salt thereof, wherein:    -   SIFA is a silicon-fluoride acceptor (SIFA) moiety which        comprises a covalent bond between a silicon and a fluorine atom        and which can be labeled with ¹⁸F by isotopic exchange between        ¹⁹F and ¹⁸F or which is labeled with ¹⁸F; preferably SIFA is the        SIFA moiety defined in claim 2;    -   m is an integer of 2 to 6, preferably 2 to 4, more preferably 2;    -   n is an integer of 2 to 6, preferably 2 to 4, more preferably 2        or 3;    -   R^(1L) is CH₂, NH or O, preferably NH;    -   R^(3L) is CH₂, NH or O, preferably NH;    -   R^(2L) is C or P(OH), preferably C;    -   X¹ is selected from an amide bond, an ether bond, a thioether        bond, an ester bond, a thioester bond, an urea bridge, and an        amine bond, preferably an amide bond;    -   X² is selected from an amide bond, an ether bond, a thioether        bond, an ester bond, a thioester bond, an urea bridge, and an        amine bond, preferably an amide bond;    -   L¹ is a divalent linking group with a structure selected from an        oligoamide, an oligoether, an oligothioether, an oligoester, an        oligothioester, an oligourea, an oligo(ether-amide), an        oligo(thioether-amide), an oligo(ester-amide), an        oligo(thioester-amide), oligo(urea-amide), an        oligo(ether-thioether), an oligo(ether-ester), an        oligo(ether-thioester), an oligo ether-urea), an        oligo(thioether-ester), an oligo(thioether-thioester), an        oligo(thioether-urea), an oligo(ester-thioester), an        oligo(ester-urea), and an oligo(thioester-urea), preferably with        a structure selected from and oligoamide and an        oligo(ester-amide);    -   X³ is selected from an amide bond, an ester bond, an ether, an        amine, and a linking group of the formula:

-   -   -   wherein the bond marked by            at the NH group is bound to R^(B) and the other bond marked            by            is bound to SIFA; preferably X³ is an amide bond;

    -   R^(B) is a trivalent coupling group;

    -   X⁴ is selected from an amide bond, an ether bond, a thioether        bond, an ester bond, a thioester bond, an urea bridge, an amine        bond, and a linking group of the formula:

wherein the amide bond marked by

is formed with the chelating group, and the other bond marked by

is bound to R^(B); preferably X⁴ is an amide bond;

-   -   R^(CH) is chelating group optionally containing a chelated        radioactive or nonradioactive cation, preferably a radioactive        or nonradioactive metal cation, preferably a chelating group as        defined in item 4, more preferably a chelating group as defined        in item 5, and most preferably a chelating group as defined in        item 6.

-   11. The conjugate in accordance with item 10, wherein L¹ comprises a    total of 1 to 5, more preferably a total of 1 to 3, and most    preferably a total of 1 or 2 amide and/or ester bonds, preferably    amide bonds, within its backbone.

-   12. The conjugate in accordance with item 10, wherein —X¹-L¹-X²—    represents one of the following structures (L-1) and (L-2):    —NH—C(O)—R⁶—C(O)—NH—R⁷—NH—C(O)—  (L-1)    —C(O)—NH—R⁸—NH—C(O)—R⁹—C(O)—NH—R¹⁰—NH—C(O)—  (L-2)    -   wherein    -   R⁶ to R¹⁰ are independently selected from C2 to C10 alkylene,        preferably linear C2 to C10 alkylene, which alkylene groups may        each be substituted by one or more substitutents independently        selected from —OH, —OCH₃, —COOH, —COOCH₃, —NH₂, and —NHC(NH)NH₂.

-   13. The conjugate in accordance with item 12, wherein the total    number of carbon atoms in R⁶ and R⁷ or R⁸ to R¹⁰, respectively, is 8    to 20, more preferably 8 to 14, without carbon atoms contained in    optional substituents.

-   14. The conjugate in accordance with 10, wherein —X¹-L¹-X²—    represents one of the following structures (L-3) and (L-4):    —NH—C(O)—R¹¹—C(O)—NH—R¹²—CH(COOH)—NH—C(O)—  (L-3)    —C(O)—NH—CH(COOH)—R¹³—NH—C(O)—R¹⁴—C(O)—NH—R¹⁵—CH(COOH)—NH—C(O)—  (L-4)    -   wherein    -   R¹¹ to R¹⁵ are independently selected from C2 to C8 alkylene,        preferably linear C2 to C8 alkylene.

-   15. The conjugate in accordance with item 14, wherein the total    number of carbon atoms in R¹¹ and R¹² or R¹³ to R¹⁵, respectively,    is 8 to 18, more preferably 8 to 12, yet more preferably 9 or 10.

-   16. The conjugate in accordance with any of items 10 to 15, wherein    R^(B) has the structure represented by formula (IV):

-   -   wherein:    -   A is selected from N, CR¹⁶, wherein R¹⁶ is H or C1-C6 alkyl, and        a 5 to 7 membered carbocyclic or heterocyclic group; preferably        A is selected from N and CH, and more preferably A is CH;    -   the bond marked by        at (CH₂)_(a) is formed with X², and a is an integer of 0 to 4,        preferably 0 or 1, and most preferably 0;    -   the bond marked by        at (CH₂)_(b) is formed with X³, and b is an integer of 0 to 4,        preferably of 0 to 2, and more preferably 0 or 1; and    -   the bond marked by        at (CH₂)_(c) is formed with X⁴, and c is an integer of 0 to 4,        preferably of 0 to 2, and more preferably 0 or 1.

-   17. The conjugate in accordance with any of items 10 to 16, which is    a compound of formula (IIIa):

-   -   or a pharmaceutically acceptable salt thereof,    -   wherein m, n, R^(1L), R^(2L), R^(3L), X¹, L¹, b, c, X⁴ and        R^(CH) are as defined in items 10 to 16.

-   18. The conjugate in accordance with item 17, wherein b+c≤1.

-   19. The conjugate in accordance with any of items 17 to 18, wherein    b+c≤3.

-   20. The conjugate in accordance with any of items 17 to 19, wherein    b is 1 and c is 0.

-   21. The conjugate in accordance with any of items 10 to 20, wherein    —X⁴—R^(CH) represents a residue of a chelating agent selected from    DOTA and DOTAGA bound with one of its carboxylic groups via an amide    bond to the remainder of the conjugate.

-   22. The conjugate in accordance with any of items 10 to 20, which is    a compound of formula (IIIb):

-   -   or a pharmaceutically acceptable salt thereof,    -   wherein    -   m, n, R^(1L), R^(2L), R^(3L), X¹, L′, b, c, X⁴ and R^(CH) are as        defined in items 10 to 20; and    -   r is 0 or 1.

-   23. The conjugate in accordance with item 22, wherein —N(H)—R^(CH)    represents a residue of a chelating agent selected from DOTA and    DOTAGA bound with one of its carboxylic groups via an amide bond to    the remainder of the conjugate.

The Figures illustrate:

FIG. 1: Exemplary correlation of the determination of the binding ofnine reference substances to Human Serum Albumin (OriginPro 2016G)

FIG. 2: Representative PET-image (maximum intensity projection, dorsalframe) of ⁶⁸Ga—^(nat)F-5 in a LNCaP tumor-bearing SCID mouse (1 h p.i.,15 min acquisition time) and ROI quantification of selected organs. Dataare expressed as mean±SD (n=4). % ID/mL=% injected dose per mL. Tumorposition is indicated by an arrow.

FIG. 3: Representative PET-image (maximum intensity projection, dorsalframe) of ⁶⁸Ga—^(nat)F-6 in a LNCaP tumor-bearing SCID mouse (1 h p.i.,15 min acquisition time) and ROI quantification of selected organs. Dataare expressed as mean±SD (n=4). % ID/mL=% injected dose per mL. Tumorposition is indicated by an arrow.

FIG. 4: Representative PET-images (maximum intensity projection, dorsalframe) of ⁶⁸Ga—^(nat)F-7 and ⁶⁸Ga—^(nat)F-7 co-injected with PMPA (8mg/kg) in LNCaP tumor-bearing SCID mice (1 h p.i., 15 min acquisitiontime) and ROI quantification of selected organs of the PET scan withoutblocking. Data are expressed as mean±SD (n=2). % ID/mL=% injected doseper mL. Tumor positions are indicated by arrows.

FIG. 5: Time activity curves (TACs, logarithmic plot) in % ID/mL derivedfrom dynamic PET data (90 min acquisition time, OSEM 3D reconstruction)in blood pool (heart), muscle, kidneys, liver and LNCaP tumor xenograftof ⁶⁸Ga—^(nat)F-7 in a LNCaP tumor-bearing SCID mouse.

FIG. 6: Representative PET-images (maximum intensity projection, dorsalframe) of ¹⁸F-7 (with free chelate) in LNCaP tumor-bearing SCID mice (1h p.i., 15 min acquisition time) and ROI quantification of selectedorgans of the PET scan without blocking. Data are expressed as mean±SD(n=3). % ID/mL=% injected dose per mL. Tumor positions are indicated byarrows.

FIG. 7: Time activity curves (TACs, logarithmic plot) in % ID/mL derivedfrom dynamic PET data (90 min acquisition time, OSEM 3D reconstruction)in blood pool (heart), muscle, kidneys, liver and LNCaP tumor xenograftof ¹⁸F-7 (with free chelate) in a LNCaP tumor-bearing SCID mouse.

FIG. 8: Biodistribution (in % ID/g) of ⁶⁸Ga—^(nat)F-7 (grey bars) and¹⁸F-7 (with free chelate) (white bars) at 1 hour p.i. in LNCaPtumor-bearing SCID mice. Data are expressed as mean±SD (n=3).

FIG. 9: Representative PET-images (maximum intensity projection, dorsalframe) of ⁶⁸Ga—^(nat)F-8 and ⁶⁸Ga—^(nat)F-8 co-injected with PMPA (8mg/kg) in LNCaP tumor-bearing SCID mice (1 h p.i., 15 min acquisitiontime) and ROI quantification of selected organs of the PET scan withoutblocking. Data are expressed as mean±SD (n=2). % ID/mL=% injected doseper mL. Tumor positions are indicated by arrows.

FIG. 10: Time activity curves (TACs, logarithmic plot) in % ID/mLderived from dynamic PET data (90 min acquisition time, OSEM 3Dreconstruction) in blood pool (heart), muscle, kidneys, liver and LNCaPtumor xenograft of ⁶⁸Ga—^(nat)F-8 in a LNCaP tumor-bearing SCID mouse.

FIG. 11: Representative PET-images (maximum intensity projection, dorsalframe) of ¹⁸F-8 (free chelate) and ¹⁸F-8 (free chelate) co-injected withPMPA (8 mg/kg) in LNCaP tumor-bearing SCID mice (1 h p.i., 15 minacquisition time) and ROI quantification of selected organs of the PETscan without blocking. Data are expressed as mean±SD (n=3). % ID/mL=%injected dose per mL. Tumor positions are indicated by arrows. Note thedifferent scaling (0-10 left; 0-20 right)

FIG. 12: Time activity curves (TACs, logarithmic plot) in % ID/mLderived from dynamic PET data (90 min acquisition time, OSEM 3Dreconstruction) in blood pool (heart), muscle, kidneys, liver and LNCaPtumor xenograft of ¹⁸F-8 in a LNCaP tumor-bearing SCID mouse.

FIG. 13: Biodistribution (in % ID/g) of ⁶⁸Ga—^(nat)F-8 (free chelate),(grey bars) and ¹⁸F-8 (white bars) at 1 hour p.i. in LNCaP tumor-bearingSCID mice. Data are expressed as mean±SD (n=3).

FIG. 14: Representative PET-images (maximum intensity projection, dorsalframe) of ⁶⁸Ga—^(nat)F-9 (free chelate) and ⁶⁸Ga—^(nat)F-9 (freechelate) co-injected with PMPA (8 mg/kg) in LNCaP tumor-bearing SCIDmice (1 h p.i., 15 min acquisition time) and ROI quantification ofselected organs of the PET scan without blocking. Data are expressed asmean±SD (n=3). % ID/mL=% injected dose per mL. Tumor positions areindicated by arrows.

FIG. 15: Time activity curves (TACs, logarithmic plot) in % ID/mLderived from dynamic PET data (90 min acquisition time, OSEM 3Dreconstruction) in blood pool (heart), muscle, kidneys, liver and LNCaPtumor xenograft of ⁶⁸Ga—^(nat)F-9 in a LNCaP tumor-bearing SCID mouse.

FIG. 16: Representative PET-images (maximum intensity projection, dorsalframe) of ¹⁸F-9 (free chelate) and ¹⁸F-9 (free chelate) co-injected withPMPA (8 mg/kg) in LNCaP tumor-bearing SCID mice (1 h p.i., 15 minacquisition time) and ROI quantification of selected organs of the PETscan without blocking. Data are expressed as mean±SD (n=4). % ID/mL=%injected dose per mL. Tumor positions are indicated by arrows.

FIG. 17: Time activity curves (TACs, logarithmic plot) in % ID/mLderived from dynamic PET data (90 min acquisition time, OSEM 3Dreconstruction) in blood pool (heart), muscle, kidneys, liver and LNCaPtumor xenograft of ¹⁸F-9 (free chelate) in a LNCaP tumor-bearing SCIDmouse.

FIG. 18: Biodistribution (in % ID/g) of ⁶⁸Ga—^(nat)F-9 (grey bars) and¹⁸F-9 (free chelate) (white bars) at 1 hour p.i. in LNCaP tumor-bearingSCID mice. Data are expressed as mean±SD (n=3).

FIG. 19: Representative PET-images (maximum intensity projection, dorsalframe) of ¹⁸F-^(nat)Ga—7 (free chelate) and ¹⁸F-^(nat)Ga—7 (freechelate) co-injected with PMPA (8 mg/kg) in LNCaP tumor-bearing SCIDmice (1 h p.i., 15 min acquisition time) and ROI quantification ofselected organs of the PET scan without blocking. Data are expressed asmean±SD (n=3). % ID/mL=% injected dose per mL. Tumor positions areindicated by arrows.

FIG. 20: Biodistribution (in % ID/g) of ¹⁸F-^(nat)Ga—7 (free chelate)(white bars), compared to the structurally identic compound⁶⁸Ga—^(nat)F-7 (free chelate) (grey bars) at 1 hour p.i. in LNCaPtumor-bearing SCID mice. Data are expressed as mean±SD (n=3).

FIG. 21: left image demonstrates the maximum intensity projection (MIR)from PET of a subject with normal biodistribution (no tumor lesionsdetectable). Images were acquired 76 min post injection of 272 MBq18F-labelled PSMA-SIFA3 (7). FIG. 21 right demonstrates the maximumintensity projection (MIP) from PET of a subject with moderatelyadvanced disease exhibiting multiple tumor lesions with highlesion-to-background ratio. Images were acquired 102 min post injectionof 312 MBq 18F-labelled PSMA-SIFA3 (7).

FIG. 22: is a graphical representation of Table 6

FIG. 23: is a graphical representation of Table 7

FIG. 24: is a graphical representation of Table 8

FIG. 25: is a graphical representation of Table 9

FIG. 26: shows: MIR (A) and transaxial images (B-D) of a 70 year oldpatient with biochemical recurrence 1.5 years after radicalprostatectomy (Gleason 8, pT2c, pN1). A single prostate cancer typicallesion with 5 mm diameter in right pelvis with high uptake of18F-labelled PSMA-SIFA3 (7) is present. Malignant nature of the lesionwas verified by histopathology.

FIG. 27: Set of images of an 80 year old patient with progressiveadvanced castration resistant prostate cancer (PSA 66.4 ng/ml). Imagesshows high uptake of 18F-labelled PSMA-SIFA3 (7) in different classes ofprostate cancer lesions (local tumor, lymph node metastases, bonemetastases, liver metastases). Lesions demonstrated are as small as 2 mm(arrows indicate representative, not all tumor lesions).

FIG. 28: shows proof of concept investigation of a ⁶⁸Ga-labelled SiFAsubstituted chelator-based PET tracer.

FIG. 29: Representative PET-images (maximum intensity projection, dorsalframe) of ¹⁸F—^(nat)Lu-rh-7 in LNCaP tumor-bearing SCID mice (1 h p.i.,15 min acquisition time) and ROI quantification of selected organs ofthe PET scan without blocking. Data are expressed as mean±SD (n=3). %ID/mL=% injected dose per mL. Tumor positions are indicated by arrows.

FIG. 30: Time activity curves (TACs, logarithmic plot) in % ID/mLderived from dynamic PET data (90 min acquisition time, OSEM 3Dreconstruction) in blood pool (heart), muscle, kidneys, liver and LNCaPtumor xenograft of ¹⁸F-^(nat)Lu-rh-7 in a LNCaP tumor-bearing SCIDmouse.

FIG. 31: Biodistribution (in % ID/g) of ¹⁷⁷Lu-^(nat)F-7, ¹⁷⁷Lu-^(nat)F-8and ¹⁷⁷Lu-^(nat)F-10 at 24 hour p.i. in LNCaP tumor-bearing SCID mice.Data are expressed as mean±SD (n=4).

FIG. 32: Biodistribution (in % ID/g) of ¹⁷⁷Lu-^(nat)F-10 at 1 hour and24 h p.i. in LNCaP tumor-bearing SCID mice. Data are expressed asmean±SD (n=4).

FIG. 33: Comparative biodistribution (in % ID/g) of established and newrhPSMA-ligands at 24 hour p.i. in LNCaP tumor-bearing SCID mice. Dataare expressed as mean±SD (n=4-5).

FIG. 34: Biodistribution (in % ID/g) of ¹⁷⁷Lu-^(nat)F-10 and⁶⁸Ga—^(nat)F-10 at 1 hour p.i. in LNCaP tumor-bearing SCID mice. Dataare expressed as mean±SD (n=4).

The Examples illustrate the invention.

EXAMPLE 1: MATERIAL AND METHODS

General

The Fmoc-(9-fluorenylmethoxycarbonyl-) and all other protected aminoacid analogs were purchased from Bachem (Bubendorf, Switzerland) or IrisBiotech (Marktredwitz, Germany). The tritylchloride polystyrene (TCP)resin was obtained from PepChem (Tübingen, Germany). Chematech (Dijon,France) delivered the chelators DOTAGA-anhydride and NOTA. The TRAPchelator(1,4,7-triazacyclononane-1,4,7-tris[methylene(2-carboxyethyl)phosphinicacid]) was synthesized as described previously (Notni et al., Chemistry(Weinheim an der Bergstrasse, Germany) 16), 7174-85 (2010)). Synthesisof the silicon fluoride acceptor SIFA-benzoic acid was performedaccording to a previously published procedure (lovkova et al., Chemistry(Weinheim an der Bergstrasse, Germany) 15, 2140-7 (2009)). All necessarysolvents and other organic reagents were purchased from either, AlfaAesar (Karlsruhe, Germany), Sigma-Aldrich (Munich, Germany) or VWR(Darmstadt, Germany). Solid phase synthesis of the peptides was carriedout by manual operation using an Intelli-Mixer syringe shaker (Neolab,Heidelberg, Germany). Analytical and preparative reversed-phase highpressure chromatography (RP-HPLC) were performed using Shimadzu gradientsystems (Shimadzu Deutschland GmbH, Neufahrn, Germany), each equippedwith a SPD-20A UV/Vis detector (220 nm, 254 nm). A Nucleosil 100 C18(125×4.6 mm, 5 μm particle size) column (CS GmbH, Langerwehe, Germany)was used for analytical measurements at a flow rate of 1 mL/min. Bothspecific gradients and the corresponding retention times t_(R) are citedin the text. Preparative HPLC purification was done with a Multospher100 RP 18 (250×10 mm, 5 μm particle size) column (CS GmbH, Langerwehe,Germany) at a constant flow rate of 5 mL/min. Analytical and preparativeradio RP-HPLC was performed using a Nucleosil 100 C18 (5 μm, 125×4.0 mm)column (CS GmbH, Langerwehe, Germany). Eluents for all HPLC operationswere water (solvent A) and acetonitrile (solvent B), both containing0.1% trifluoroacetic acid. Electrospray ionization-mass spectra forcharacterization of the substances were acquired on an expression^(L)CMS mass spectrometer (Advion Ltd., Harlow, UK). Radioactivity wasdetected through connection of the outlet of the UV-photometer to aNaI(TI) well-type scintillation counter from EG&G Ortec (Munich,Germany). Gel permeation chromatography (GPC) was done on Sephadex GP-10(100 g, bed size approx. 30×3 cm) with water as eluent, separating theeluate in 20 mL fractions. NMR spectra were recorded on Bruker AVHD-300or AVHD-400 spectrometers at 300 K. pH values were measured with aSevenEasy pH-meter (Mettler Toledo, Gießen, Germany).

Synthesis Protocols

1) Solid-Phase Peptide Synthesis Following the Fmoc-Strategy

TCP-Resin Loading

Loading of the tritylchloride polystyrene (TCP) resin with aFmoc-protected amino acid (AA) was carried out by stirring a solution ofthe TCP-resin (1.95 mmol/g) and Fmoc-AA-OH (1.5 eq.) in anhydrous DCMwith DIPEA (4.5 eq.) at room temperature for 2 h. Remainingtritylchloride was capped by the addition of methanol (2 mL/g resin) for15 min. Subsequently the resin was filtered and washed with DCM (2×5mL/g resin), DMF (2×5 mL/g resin), methanol (5 mL/g resin) and dried invacuo. Final loading/of Fmoc-AA-OH was determined by the followingequation:

${l\mspace{11mu}\left\lbrack \frac{mmol}{g} \right\rbrack} = \frac{\left( {m_{2} - m_{1}} \right) \times 1000}{\left( {M_{W} - M_{HCl}} \right)m_{2}}$

-   -   m₂=mass of loaded resin [g]    -   m₁=mass of unloaded resin [g]    -   M_(W)=molecular weight of AA [g/mol]    -   M_(HCl)=molecular weight of HCl [g/mol]        On-Resin Peptide Formation

The respective side-chain protected Fmoc-AA-OH (1.5 eq.) was dissolvedin DMF (8 mL/g resin) and pre-activated by adding TBTU (1.5 eq.), HOBt(1.5 eq.) and DIPEA (4.5 eq.). Pre-activation for SIFA-BA was performedanalogously. For azido-substituted amino acids (2.0 eq.), HATU (3.0eq.), HOAt (3.0 eq.) and DIPEA (6.0 eq.) were used. After activation for15 minutes, the solution was added to resin-bound free amine peptideTCP-AA-NH₂ and shaken for 2 h at room temperature. Subsequently, theresin was washed with DMF (6×5 mL/g resin) and after Fmoc-deprotectionthe next amino acid was coupled analogously.

On-Resin Fmoc-Deprotection

The resin-bound Fmoc-peptide was treated with 20% piperidine in DMF(v/v, 8 mL/g resin) for 5 min and subsequently for 15 min. Afterwards,the resin was washed thoroughly with DMF (8×5 mL/g resin).

On-Resin Dde-Deprotection

The Dde-protected peptide (1.0 eq.) was dissolved in a solution of 2%hydrazine monohydrate in DMF (v/v, 5 mL/g resin) and shaken for 15 min.In the case of present Fmoc-groups, Dde-deprotection was performed byadding a solution of imidazole (0.46 g), hydroxylamine hydrochloride(0.63 g) in NMP (2.5 mL) and DMF (0.5 mL) for 3 h at room temperature.After deprotection the resin was washed with DMF (6×5 mL/g resin).

On-Resin Alloc-Deprotection

The allyloxy-protecting group was removed by the addition oftriisopropylsilane (TIPS) (50.0 eq.) andtetrakis(triphenylphosphine)palladium(0) (Pd(PPh₃)₄) (0.3 eq.) dissolvedin DCM (6 mL). After 1.5 h at room temperature, the resin was washedwith DCM (6×5 mL/g resin).

tBu/Boc Deprotection

Removal of tBu/tBoc-protecting groups was carried out by dissolving thecrude product in TFA and stirring for 40 min at RT. After removing TFAunder a stream of nitrogen, the residue was dissolved in a mixture oftert-butanol and water. After lyophilisation the crude peptide wasobtained.

Peptide Cleavage from the Resin

-   -   a) Preservation of acid labile protecting groups: The        resin-bound peptide was dissolved in a mixture of DCM/TFE/AcOH        (v/v/v; 6/3/1, 8 mL/g resin) and shaken for 30 min. The solution        containing the fully protected peptide was filtered off and the        resin was treated with another portion of the cleavage solution        for 30 min. Both fractions were combined and acetic acid was        removed under reduced pressure by successively adding toluene        and water. After lyophilisation of remaining water, the crude        fully protected peptide was obtained.    -   b) Deprotection of all acid labile protecting groups: The fully        protected resin-bound peptide was dissolved in a mixture of        TFA/TIPS/water (v/v/v; 95/2.5/2.5) and shaken for 30 min. The        solution was filtered off and the resin was treated in the same        way for another 30 min. Both filtrates were combined and        concentrated under a stream of nitrogen. After dissolving the        residue in a mixture of tert-butanol and water and subsequent        lyophilisation the crude peptide was obtained.

2) Synthesis of the Binding Motifs Lys-urea-Glu ((tBuO)KuE(OtBu)₂) (1)

The synthesis of the tBu-protected Lys-urea-Glu binding motif (EuK) wascarried out as described previously by solution phase synthesis(Weineisen et al., EJNMMI research 4, 63 (2014)). The product wasobtained as a waxy solid (91.5%). HPLC (10 to 90% B in 15 min):t_(R)=12.6 min. Calculated monoisotopic mass (C₂₄H₄₅N₃O₇): 487.6 found:m/z=488.3 [M+H]⁺, 510.3 [M+Na]⁺.

Glu-urea-Glu ((tBuO)EuE(OtBu)₂) (2)

The tBu-protected Glu-urea-Glu binding motif (EuE) was synthesizedaccording to a previously published procedure (Weineisen et al., EJNMMIresearch 4, 63 (2014)). The product was obtained as a hygroscopic solid(84%). HPLC (10% to 90% B in 15 min): t_(R)=11.3 min. Calculatedmonoisotopic mass (C₂₃H₄₉N₂O₉): 488.3; found: m/z=489.4 [M+H]⁺, 516.4[M+Na]⁺.

PfpO-Sub-(tBuO)KuE(OtBu)₂ (3)

Conjugation of EuK to the suberic acid spacer was performed as describedpreviously (Weineisen et al., EJNMMI research 4, 63 (2014)). The productwas obtained as a colorless oil (72%). HPLC (10 to 90% B in 15 min):t_(R)=15.5 min. Calculated monoisotopic mass (C₃₈H₅₆F₅N₃O₁₀): 809.4found: m/z=810.6 [M+H]⁺, 832.4 [M+Na]⁺.

Conjugation of the EuK-Sub-Moiety (3) to the Peptide

The N-terminal deprotected peptide (1.0 eq.) was added to a solution of3 (1.2 eq.) in DMF and TEA (8 eq.) was added. After stirring thesolution for 2 h at room temperature, DMF was removed in vacuo. Forcleavage of the tBu-esters, TFA was added and the solution was stirredfor 45 min at room temperature. After removing TFA under a stream ofnitrogen, the crude product was purified by RP-HPLC.

3) Synthesis of Propargyl-TRAP (4)

Synthesis of propargyl-TRAP was carried out as described previously(Reich et al., Chemical communications (Cambridge, England) 53,2586-2589(2017)). Final purification was done by preparative HPLC,affording 60.2 mg (82.4 μmol, 46%) of propargyl-TRAP as a colorlesssolid. HPLC (2 to 40% B in 20 min): t_(R)=6.0 min. Calculatedmonoisotopic mass (C₂₁H₃₉N₄O₁₁P₃): 616.5 found: m/z=617.5 [M+H]⁺.

¹H-NMR (300 MHz, D₂O, 300 K) δ=1.96-2.08 (m, 6H, C(O)—CH₂), 2.46-2.55(m, 2H, P^(B)—CH₂—C), 2.59-2.70 (m, 4H, P^(A)—CH₂—C), 3.36 (d, 2H,²J_(PH)=6 Hz, P^(B)—CH₂—N), 3.41 (d, 4H, ²J_(PH)=6 Hz, P^(A)—CH₂—N),3.47-3.48 (m, 12H, ring-CH₂), 3.95 (d, 2H, CH₂—C≡CH, ⁴J_(HH)=3 Hz) ppm*.¹³C{¹H}-NMR (101 MHz, D₂O, 300 K) δ=24.61 (d, ¹J_(PP)=95 Hz, P^(B)—C—C),25.07 (d, ¹J_(PP)=94 Hz, P^(A)—C—C), 26.71 (d, ²J_(PP)=4 Hz, P^(B)—C—C),27.82 (d, ²J_(PP)=3 Hz, P^(A)—C—C), 28.89 (C—C≡C), 51.29/51.41/51.50(three different ring-C), 53.66 (d, ¹J_(PP)=91 Hz, N—C—P^(A)), 53.66 (d,¹J_(PP)=90 Hz, N—C—P^(B)), 71.79 (C—C≡C), 79.65 (C—C≡C), 174.46 (d,³J_(PP)=14 Hz, N(H)—C═O^(B)), 177.24 (d, ³J_(PP)=13 Hz, C═O^(A)) ppm*.³¹P{¹H}-NMR (162 MHz, D₂O, 300 K) δ=37.99 (P^(A)), 38.68 (P^(B)) ppm*.*: indices ^(A) and ^(B) indicate P and O atoms belonging to theundecorated^(A) and decorated^(B) side arm, respectively.

Coupling of Propargyl-TRAP (4) to the Peptide

For conjugation of azide-functionalized peptides to propargyl-TRAP viacopper(I)-catalyzed alkyne-azide cycloaddition a previously developedprocedure was applied (Reich et al., Chemical communications (Cambridge,England) 53, 2586-2589(2017)). Briefly, propargyl-TRAP (1.0 eq.) wasdissolved in water (40 mM solution) and combined with a solution of thepeptide (1.1 eq.) in a 1:1 (v/v) mixture of tBuOH and water.Subsequently, a solution of sodium ascorbate (0.5 M, 50 eq.) in waterwas added. In order to start the reaction, an aqueous solution ofCu(OAc)₂.H₂O (0.05 M, 1.2 eq.) was added, which resulted in a brownprecipitate that dissolved after stirring in a clear green solution. Fordemetallation of TRAP, an aqueous solution of1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA, 8 mM, 12 eq.) wasadded and the pH was adjusted to 2.2 with 1 M aq. HCl. After either 1 hat 60° C., or 48 h at room temperature the mixture was directlysubjected to preparative HPLC purification.

4) Conjugation of DOTAGA

The condensation of peptides and the respective chelatorDOTAGA-anhydride are described in several publications and summarized asfollows: The N-terminal deprotected peptide (1.0 eq.) was dissolvedtogether with DOTAGA-anhydride (1.5 eq.) and DIPEA (10.0 eq.) in dryDMF. After stirring the reaction mixture overnight, DMF was removed invacuo, yielding the crude product.

5) Synthesis of EuK-Based PSMA-SIFA Inhibitors PSMA-SIFA1 (5)

PSMA-SIFA1 was synthesized according to standard Fmoc-solid-phasepeptide synthesis (SPPS) on a tritylchloride polystyrene (TCP) resin,applying the above mentioned methods. Briefly, resin boundFmoc-D-Lys(Boc) was Fmoc deprotected with 20% piperidine in DMF andFmoc-D-Dap(Dde)-OH (2.0 eq.) was conjugated applying HOBt (2.0 eq.),TBTU (2.0 eq.) and DIPEA (6.0 eq.) in DMF. After orthogonalDde-deprotection with imidazole and hydroxylamine hydrochloride in amixture of NMP and DMF, SIFA-BA (1.5 eq.) was conjugated analogously.Subsequent Fmoc-deprotection and mild cleavage from the resin with TFEand AcOH in DCM yielded the Boc-protected peptide backbone. Condensationof DOTAGA-anhydrid (1.5 eq.) was performed by adding DIPEA (10 eq.) inDMF. After Boc-deprotection in TFA, the PfpO-Sub-(tBuO)KuE(OtBu)₂ moiety(1.2 eq.) was added in a mixture of TEA (8 eq.) and DMF. Final cleavageof the tBu-esters in TFA and RP-HPLC purification yielded PSMA-SIFA1(70%) as a colorless solid. HPLC (10 to 90% B in 15 min): t_(R)=9.1 min.Calculated monoisotopic mass (C₆₃H₁₀₂FN₁₁O₂₂Si): 1411.7 found:m/z=1412.3 [M+H]⁺, 706.8 [M+2H]²⁺.

Synthesis of PSMA-SIFA2 was carried out by applying the general methodsand procedures mentioned before. Shortly, resin bound Fmoc-D-Lys(Boc)-OHwas Fmoc-deprotected with 20% piperidine in DMF and conjugated toN₃-L-Dap(Fmoc)-OH (2.0 eq.) with HATU (3.0 eq.), HOAt (3.0 eq.) andDIPEA (6.0 eq.) in DMF. After cleavage of the Fmoc-group, SIFA-BA (1.5eq.) was added with HOBt (1.5 eq.), TBTU (1.5 eq.) and DIPEA (4.5 eq.)in DMF. Subsequent cleavage from the resin with TFA yielded the fullydeprotected peptide backbone. For conjugation of the EuK-moiety,PfpO-Sub-(tBuO)KuE(OtBu)₂ (1.2 eq.) was added in a mixture of TEA (8eq.) and DMF. Cleavage of the tBu-esters was performed by adding TFA. Ina final step the purified peptide (1.1 eq.) was reacted withpropargyl-TRAP (1.0 eq.) in a copper(I)-catalyzed alkyne-azidecycloaddition, as mentioned above. After RP-HPLC purification PSMA-SIFA2(4%) was obtained as a colourless solid. HPLC (10 to 90% B in 15 min):t_(R)=8.5 min. Calculated monoisotopic mass (C₆₅H₁₀₉FN₁₃O₂₄P₃Si): 1595.7found: m/z=1596.5 [M+H]⁺, 799.1 [M+2H]²⁺.

6) Synthesis of EuE-Based PSMA-SIFA Inhibitors

PSMA-SIFA3 was synthesized applying the standard Fmoc-SPPS protocoldescribed above. Briefly, resin bound Fmoc-D-Orn(Dde)-OH wasFmoc-deprotected with 20% piperidine in DMF and (tBuO)EuE(OtBu)₂ (2.0eq.) was conjugated with HOBt (2.0 eq.), TBTU (2.0 eq.) and DIPEA (6.0eq.) in DMF. After cleavage of the Dde-group with a mixture of 2%hydrazine in DMF, a solution of succinic anhydride (7.0 eq.) and DIPEA(7.0 eq.) in DMF was added. Conjugation of Fmoc-D-Lys-OAII.HCl (1.5 eq.)was achieved by adding a mixture of HOBt (1.5 eq.), TBTU (1.5 eq.) andDIPEA (4.5 eq.) in DMF. After cleavage of the Fmoc-group with 20%piperidine in DMF, the free amine was conjugated to Fmoc-D-Dap(Dde)-OH(2.0 eq.) after pre-activation of the amino acid in a mixture of HOBt(2.0 eq.), TBTU (2.0 eq.) and DIPEA (6.0 eq.) in DMF. Followingorthogonal Dde-deprotection was done using imidazole and hydroxylaminehydrochloride dissolved in a mixture of NMP and DMF. SIFA-BA (1.5 eq.)was reacted with the free amine of the side chain with HOBt (1.5 eq.),TBTU (1.5 eq.) and DIPEA (4.5 eq.) as activation reagents in DMF. Theallyloxy-protecting group was removed by the addition of TIPS (50.0 eq.)and Pd(PPh₃)₄ (0.3 eq.) dissolved in DCM. After Fmoc-deprotection withpiperidine, the peptide was cleaved from the resin with preservation ofthe acid labile protecting groups by using a mixture of TFE and AcOH inDCM. Final condensation of DOTAGA-anhydrid (1.5 eq.) was achieved withpiperidine (10 eq.) in DMF. After cleavage of the tBu-esters of theEuE-moiety with TFA, the crude peptide was purified by RP-HPLC, yieldingPSMA-SIFA3 (24%) as a colorless solid. HPLC (10 to 70% B in 15 min):t_(R)=10.4 min. Calculated monoisotopic mass (C₆₃H₉₉FN₁₂O₂₅Si): 1470.7found: m/z=1471.8 [M+H]⁺, 736.7 [M+2H]²⁺.

HOBt, TBTU, DIPEA (DMF); i) TIPS, Pd(PPh₃)₄, (DCM); j) TFE, AcOH (DCM);k) DOTAGA-anhydrid, DIPEA, (DMF); I) TFA.

PSMA-SIFA4 was synthesized through SPPS as compound 7, with onedeviation; After conjugation of Fmoc-D-Dap(Dde)-OH, the Fmoc-protectinggroup was cleaved first with 20% piperidine in DMF and SIFA-BA wasreacted with the free N-terminus of the peptide. The remaining Dde-groupwas cleaved after removing the allyloxy-protecting group by usingimidazole and hydroxylamine hydrochloride dissolved in a mixture of NMPand DMF. Following reaction steps were identical to PSMA-SIFA3. AfterRP-HPLC purification, PSMA-SIFA4 (11%) was obtained as a colorlesssolid. HPLC (10 to 70% B in 15 min): t_(R)=10.4 min. Calculatedmonoisotopic mass (C₆₃H₉₉FN₁₂O₂₅Si): 1470.7 found: m/z=1471.7 [M+H]⁺,736.8 [M+2H]²⁺.

The peptide backbone of PSMA-SIFA5 was prepared analogously to ligand 7and 8. A difference was the use of N₃-L-Dap(Fmoc)-OH instead ofFmoc-D-Dap(Dde)-OH, which was required for the final click reaction withpropargyl-TRAP. The azido-substituted amino acid (2.0 eq.) wasconjugated with HATU (3.0 eq.), HOAt (3.0 eq.) and DIPEA (6.0 eq.) inDMF. After Fmoc-deprotection of its side chain with 20% piperidine,SIFA-BA (1.5 eq.) was reacted as mentioned above. Until conjugation ofthe chelator moiety, all remaining reaction steps were identical topeptide 7 and 8. After cleavage from the resin with TFA under concurrentdeprotection of all acid labile protecting groups, the purifiedEuE-azido-conjugate (1.1 eq.) was reacted with propargyl-TRAP (1.0 eq.)in a copper(I)-catalyzed alkyne-azide cycloaddition. RP-HPLCpurification yielded PSMA-SIFA5 (9%) as a colourless solid. HPLC (10 to90% B in 15 min): t_(R)=8.7 min. Calculated monoisotopic mass(C₆₅H₁₀₆FN₁₄O₂₇P₃Si): 1654.6 found: m/z=1655.6 [M+H]⁺, 828.4 [M+2H]²⁺.

Synthesis of ^(nat)Ga-TRAP complexes: 500 μL of a 2 mM stock solution ofthe PSMA precursor in DMSO was combined with 750 μL of a 2 mM Ga(NO₃)₃solution in water. Complexation occurred instantaneously at roomtemperature. Completeness of the reaction was controlled by RP-HPLC andsubsequent mass spectrometry.

^(nat)Ga-PSMA-SIFA2: HPLC (10 to 90% B in 15 min): t_(R)=9.5 min.Calculated monoisotopic mass (C₆₅H₁₀₆FGaN₁₃O₂₄P₃Si): 1661.6 found:m/z=1663.9 [M+H]⁺, 832.6 [M+2H]²⁺.

^(nat)Ga-PSMA-SIFA5: HPLC (10 to 90% B in 15 min): t_(R)=9.0 min.Calculated monoisotopic mass (C₆₅H₁₀₃FGaN₁₄O₂₇P₃Si): 1720.5 found:m/z=1720.8 [M+H]⁺, 861.1 [M+2H]²⁺.

Synthesis of ^(nat)Ga-DOTAGA complexes: 500 μL of a 2 mM stock solutionof the PSMA precursor in DMSO was combined with 1500 μL of a 2 mMGa(NO₃)₃ solution in water. The reaction mixture was heated for 30 minat 60° C. Outcome of the reaction was monitored by RP-HPLC andsubsequent mass spectrometry. For radioactive labelling of[^(nat)Ga]PSMA-SIFA ligands with ¹⁸F, the complexed compound waspurified by RP-HPLC before the labelling reaction.

^(nat)Ga-PSMA-SIFA1: HPLC (10 to 90% B in 15 min): t_(R)=9.1 min.Calculated monoisotopic mass (C₆₃H₉₉FGaN₁₁O₂₂Si): 1477.6 found:m/z=1479.5 [M+H]⁺, 740.2 [M+2H]²⁺.

^(nat)Ga-PSMA-SIFA3: HPLC (10 to 70% B in 15 min): t_(R)=10.4 min.Calculated monoisotopic mass (C₆₃H₉₆FGaN₁₂O₂₅Si): 1536.6 found:m/z=1539.4 [M+H]⁺, 770.3 [M+2H]²⁺.

^(nat)Ga-PSMA-SIFA4: HPLC (10 to 70% B in 15 min): HPLC (10 to 70% B in15 min): t_(R)=10.4 min. Calculated monoisotopic mass(C₆₃H₉₆FGaN₁₂O₂₅Si): 1536.6 found: m/z=1539.1 [M+H]⁺, 770.5 [M+2H]²⁺.

^(nat)Lu-complexation: The corresponding ^(nat)Lu^(III)-complexes wereprepared from a 2 mM aqueous solution of the PSMA inhibitor with a 2.5molar excess of LuCl₃ (20 mM solution), heated to 95° C. for 30 min.After cooling, the ^(nat)Lu^(III)-chelate formation was confirmed usingHPLC and MS. The resulting 1 mM aqueous solutions of the respective^(nat)Lu-complexes were then diluted and used in the in vitro IC₅₀studies without further processing.

Radiolabelling

⁶⁸Ga-labelling: ⁶⁸Ga-labelling was done using an automated system(GallElut⁺ by Scintomics, Germany) as described previously (Notni etal., EJNMMI research, 28 (2012)). Briefly, the ⁶⁸Ge/⁶⁸Ga-generator withSnO₂ matrix (IThemba LABS) was eluted with 1.0 M aqueous HCl, from whicha fraction (1.25 mL) of approximately 80% of the activity (500-700 MBq),was transferred into a reaction vial (ALLTECH, 5 mL). The reactor wasloaded before elution with 2-5 nmol of respective chelator conjugate inan aqueous 2.7 M HEPES solution (DOTAGA-conjugates: 900 μL,TRAP-conjugates: 400 μL). After elution the vial was heated for 5minutes at 95° C. Purification was done by passing the reaction mixtureover a solid phase extraction cartridge (C 8 light, SepPak), which waspurged with water (10 mL) and the product eluted with 50% aqueousethanol (2 mL), phosphate buffered saline (PBS, 1 mL) and again water (1mL). After removing ethanol in vacuo, purity of the radiolabelledcompounds was determined by radio-TLC (ITLC-SG chromatography paper,mobile phase: 0.1 M trisodium citrate and 1:1 mixture (v/v) of 1 Mammonium acetate and methanol).

¹⁸F-labelling: For ¹⁸F-labelling a previously published procedure(Wangler et al., Nat Protoc 7, 1946-55 (2012)). was applied, which wasslightly modified. Briefly, aqueous ¹⁸F⁻ was passed through a SAXcartridge (Sep-Pak Accell Plus QMA Carbonate light), which waspreconditioned with 10 mL of water. After drying with 10 mL of air,water was removed, by rinsing the cartridge with 10 mL of anhydrousacetonitrile followed by 20 mL of air. ¹⁸F was eluted with 100 μmol of[K⁺⊂2.2.2]OH⁻ dissolved in 500 μL of anhydrous acetonitrile. Beforelabelling, 25 μmol of oxalic acid in anhydrous acetonitrile (1 M, 25 μL)were added. This mixture was used as a whole or aliquot for fluorinationof 10-25 μmol of PSMA-SIFA (1 M in anhydrous DMSO). The resultingreaction mixture was incubated for 5 minutes at room temperature. Forpurification of the tracer, a Sep-Pak C18 light cartridge,preconditioned with 10 mL EtOH, followed by 10 mL of H₂O was used. Thelabelling mixture was diluted with 9 mL 0.1 M HEPES buffer (pH 3) andpassed through the cartridge followed by 10 mL of H₂O. The peptide waseluted with 500 μL of a 4:1 mixture (v/v) of EtOH in water.Radiochemical purity of the labelled compound was determined by radioRP-HPLC and radio-TLC (Silica gel 60 RP-18 F₂₅₄S, mobile phase: 3:2mixture (v/v) of MeCN in H₂O supplemented with 10% of 2 M NaOAc solutionand 1% of TFA).

¹²⁵I-labelling: The reference ligand for in vitro studies([¹²⁵I]I-BA)KuE was prepared according to a previously publishedprocedure (Weineisen et al., EJNMMI research, 4, 63 (2014)). Briefly,0.1 mg of the stannylated precursor (SnBu₃-BA)(OtBu)KuE(OtBu)₂ wasdissolved in a solution containing 20 μL peracetic acid, 5.0 μL (21 MBq)[¹²⁵I]NaI (74 TBq/mmol, 3.1 GBq/mL, 40 mM NaOH, Hartmann Analytic,Braunschweig, Germany), 20 μL MeCN and 10 μL acetic acid. The reactionsolution was incubated for 10 min at RT, loaded on a cartridge andrinsed with 10 mL water (C18 Sep Pak Plus cartridge, preconditioned with10 mL MeOH and 10 mL water). After elution with 2.0 mL of a 1:1 mix(v/v) of EtOH/MeCN, the radioactive solution was evaporated to drynessunder a gentle nitrogen stream and treated with 200 μL TFA for 30 minwith subsequent evaporation of TFA. The crude product of ([¹²⁵I]I-BA)KuEwas purified by RP-HPLC (20% to 40% B in 20 min): t_(R)=13.0 min.

¹⁷⁷Lu-labeling: To a reaction volume of 10 μL 1.0 M NH₄OAc buffer pH=5.9was added 0.75 to 1.0 nmol tracer (7.5 to 10 μL), 10 to 40 MBq ¹⁷⁷LuCl₃(Specific Activity (A_(S))>3000 GBq/mg, 740 MBq/mL, 0.04 M HCl, ITG,Garching, Germany) and finally filled up to 100 μL with trace-pure Water(Merck, Darmstadt, Germany). The reaction mixture was heated for 30 minat 95° C. and the radiochemical purity was determined using radio-TLC(Silica gel 60 RP-18 F₂₅₄s, 3:2 mixture (v/v) of MeCN in H₂Osupplemented with 10% of 2 M NaOAc solution and 1% of TFA, R_(f)=0.5).

In Vitro Experiments

Determination of IC₅₀

The PSMA-positive LNCaP cells were grown in Dublecco modified Eaglemedium/Nutrition Mixture F-12 with Glutamax-I (1:1) (Invitrigon),supplemented with 10% fetal calf serum and maintained at 37° C. in ahumidified 5% CO₂ atmosphere. For determination of the PSMA affinity(IC₅₀), cells were harvested 24±2 hours before the experiment and seededin 24-well plates (1.5×10⁵ cells in 1 mL/well). After removal of theculture medium, the cells were treated once with 500 μL of HBSS (Hank'sbalanced salt solution, Biochrom, Berlin, Germany, with addition of 1%bovine serum albumin (BSA)) and left 15 min on ice for equilibration in200 μL HBSS (1% BSA). Next, 25 μL per well of solutions, containingeither HBSS (1% BSA, control) or the respective ligand in increasingconcentration (10⁻¹⁰-10⁻⁴ M in HBSS, were added with subsequent additionof 25 μL of ([¹²⁵I]I-BA)KuE (2.0 nM) in HBSS (1% BSA). All experimentswere performed at least three times for each concentration. After 60 minincubation on ice, the experiment was terminated by removal of themedium and consecutive rinsing with 200 μL of HBSS. The media of bothsteps were combined in one fraction and represent the amount of freeradioligand. Afterwards, the cells were lysed with 250 μL of 1 M NaOHand united with the 200 μL HBSS of the following wash step.Quantification of bound and free radioligand was accomplished in aγ-counter.

Internalization

For internalization studies, LNCaP cells were harvested 24±2 hoursbefore the experiment and seeded in 24-well plates (1.25×10⁵ cells in 1mL/well). Subsequent to the removal of the culture medium, the cellswere washed once with 500 μL DMEM-F12 (5% BSA) and left to equilibratefor at least 15 min at 37° C. in 200 μL DMEM-F12 (5% BSA). Each well wastreated with either 25 μL of either DMEM-F12 (5% BSA) or a 100 μM PMPAsolution for blockade. Next, 25 μL of the ⁶⁸Ga/¹⁸F-labelled PSMAinhibitor (5.0 nM) was added and the cells incubated at 37° C. for 60min. The experiment was terminated by placing the 24-well plate on icefor 3 min and consecutive removal of the medium. Each well was rinsedwith 250 μL HBSS and the fractions from these first two steps combined,representing the amount of free radioligand. Removal of surface boundactivity was accomplished by incubation of the cells with 250 μL ofice-cold PMPA (10 μM in PBS) solution for 5 min and rinsed again withanother 250 μL of ice-cold PBS. The internalized activity was determinedby incubation of the cells in 250 μL 1 M NaOH and the combination withthe fraction of a subsequent wash step with 250 μL 1.0 M NaOH. Eachexperiment (control and blockade) was performed in triplicate. Free,surface bound and internalized activity was quantified in a γ-counter.All internalization studies were accompanied by reference studies using([¹²⁵I]I-BA)KuE (c=0.2 nM), which were performed analogously. Data werecorrected for non-specific internalization and normalized to thespecific-internalization observed for the radioiodinated referencecompound.

Octanol-Water Partition Coefficient

Approximately 1 MBq of the labelled tracer was dissolved in 1 mL of a1:1 mixture (by volumes) of phosphate buffered saline (PBS, pH 7.4) andn-octanol in an Eppendorf tube. After vigorous mixing of the suspensionfor 3 minutes at room temperature, the vial was centrifuged at 15000 gfor 3 minutes (Biofuge 15, Heraus Sepatech, Osterode, Germany) and 100μL aliquots of both layers were measured in a gamma counter. Theexperiment was repeated at least six times.

HSA Binding

For the determination of HSA binding, a Chiralpak HSA column (50×3 mm, 5μm, H₁₃H-2433) was used at a constant flow rate of 0.5 mL/min. Themobile phase (A: NH₄OAc, 50 mM in water, pH 7 and B: isopropanol) wasfreshly prepared for each experiment and only used for one day. Thecolumn was kept at room temperature and each run was stopped afterdetection of the signal to reduce the acquisition time. All substanceswere dissolved in a 0.5 mg/ml concentration in 50% 2-propanol and 50% 50mM pH 6.9 ammonium acetate buffer. The chosen reference substancesdisplay a range of HSA binding from 13% to 99% since a broad variety ofalbumin binding regarding the peptides was assumed. All nine referencesubstances (see Table 1) were injected consecutively to establish anon-linear regression with OriginPro 2016G; see FIG. 1.

TABLE 1 Reference substances (Yamazaki et al., Journal of pharmaceuticalsciences 93, 1480-94 (2004)) used for the calibration of the HSA-column.Reference t_(R) Log t_(R) Lit. HSA % Log K HSA p-benzylalcohol 2.40 0.3813.15 −0.82 Aniline 2.72 0.43 14.06 −0.79 Phenol 3.28 0.52 20.69 −0.59Benzoic Acid 4.08 0.61 34.27 −0.29 Carbamazepine 4.15 0.62 75.00 0.46p-nitrophenol 5.62 0.75 77.65 0.52 Estradiol 8.15 0.91 94.81 1.19Probenecid 8.84 0.95 95.00 1.20 Glibenclamide 29.18 1.47 99.00 1.69

The retention time is shown exemplary for a conducted experiment; t_(R)retention time; Lit. HSA literature value of human serum albumin bindingin [%]; Log K HAS logarithmic K of human serum albumin binding.

In Vivo Experiments

All animal experiments were conducted in accordance with general animalwelfare regulations in Germany and the institutional guidelines for thecare and use of animals. To establish tumor xenografts, LNCaP cells (10⁷cells/200 μL) were suspended in a 1:1 mixture (v/v) of Dulbecco modifiedEagle medium/Nutrition Mixture F-12 with Glutamax-I (1:1) and Matrigel(BD Biosciences, Germany), and inoculated subcutaneously onto the rightshoulder of 6-8 weeks old CB17-SCID mice (Charles River, Sulzfeld,Germany). Mice were used for when tumors had grown to a diameter of 5-8mm (3-4 weeks after inoculation).

μPET Imaging

Imaging studies were performed at a Siemens Inveon small-animal PETsystem. Data were reconstructed as single frames employing athree-dimensional ordered subset expectation maximum (OSEM3D) algorithm,followed by data analysis (ROI-based quantification) using InveonResearch Workplace software. For PET studies mice were anesthetized withisoflurane and injected with 0.15-0.25 nmol (2-20 MBq) of the ⁶⁸Ga or¹⁸F-labelled tracer into the tail vein. Dynamic imaging was performedafter on-bed injection for 90 minutes. Static images were recorded onehour after the injection with an acquisition time of 15 minutes. Forblockade 8 mg/kg of PMPA was administered directly before tracerinjection.

Biodistribution

Approximately 2-20 MBq (0.2 nmol) of the ⁶⁸Ga or ¹⁸F-labelled PSMAinhibitors were injected into the tail vein of LNCaP tumor-bearing maleCB-17 SCID mice and sacrificed after 1 h post injection (n=3). Selectedorgans were removed, weighted and measured in a γ-counter.

In Human Experiments

A proof-of-concept evaluation of use in humans was conducted undercompassionate use. The agent was applied in compliance with The GermanMedicinal Products Act, AMG § 13 2b, and in accordance with theresponsible regulatory body (Government of Oberbayern).

All subjects were examined on a Biograph mCT scanner (Siemens MedicalSolutions, Erlangen, Germany) or a Biograph mMR scanner (Siemens MedicalSolutions, Erlangen, Germany). All PET scans were acquired in 3D-modewith an acquisition time of 2-4 min per bed position. Emission data werecorrected for randoms, dead time, scatter, and attenuation and werereconstructed iteratively by an ordered-subsets expectation maximizationalgorithm (four iterations, eight subsets) followed by apostreconstruction smoothing Gaussian filter (5-mm full width atone-half maximum). Images in 53 subjects with prostate cancer wereobtained after injection of a mean of 324 (range 236-424) MBq18F-labelled PSMA-SIFA3 (7) at a mean of 84 (range 42-166) min postinjection. 47 subjects underwent imaging on a PET/CT, 6 subjects on aPET/MR scanner. In 33 subjects furosemide was applied at the time oftracer injection, in 20 subjects no furosemide was given.

The mean and maximum standardized uptake values (SUVmean/SUVmax) ofparotid glands, submandibular glands, lungs, mediastinal blood pool,liver, spleen, pancreas head, duodenum, kidneys, bladder andnon-diseased bone were analysed. For calculation of the SUV, circularregions of interest were drawn around areas with focally increaseduptake in transaxial slices and automatically adapted to athree-dimensional volume of interest (VOI) at a 50% isocontour. Lesionsthat were visually considered as suggestive of relapses or metastases ofprostate cancer were counted. One or two lesions from the same type(local tumor, lymph node metastases, bone metastases, visceralmetastases) were analysed per patient using SUVmax and SUVmean asdescribed above. Gluteal muscle served as background.

EXAMPLE 2: RESULTS

Overview of the Synthesised PSMA-SIFA Ligands

Lipophilicities

The determined octanol/water partition coefficients (log D) of the ⁶⁸Ga—or ¹⁸F-labelled compounds are presented in Table 2. Within the⁶⁸Ga-labelled EuK-based inhibitors, the TRAP-functionalized compound(5), was found to be more hydrophilic than 6, where DOTAGA was used as achelator. This result was also found for the ⁶⁸Ga-labelled EuE-basedagents, where the TRAP-derivative (9) showed the highest lipophilicity.All of the ¹⁸F-labelled compounds showed lower hydrophilicities,compared to the ⁶⁸Ga-labelled tracers.

TABLE 2 log D values of the synthesized radiolabelled PSMA-SIFA ligands(n = 6). log D log D log D ligand ⁶⁸Ga-^(nat)F-L ¹⁸F-L ¹⁷⁷Lu-^(nat)F-L 5−2.75 ± 0.07 n.d. 6 −3.00 ± 0.09 n.d. 7 −3.18 ± 0.05 −1.98 ± 0.04 −4.20± 0.09 8 −2.59 ± 0.04 −2.25 ± 0.07 −3.75 ± 0.06 9 −3.26 ± 0.06 −2.23 ±0.07 10 −3.59 ± 0.05 11 −3.62 ± 0.06Determination of PSMA Affinities

The synthesized compounds bearing a EuE-binding motif (7, 8, 9) showedhigher PSMA affinities compared to the EuK-based agents (5, 6).Compounds with a TRAP-chelator (6 and 9) showed slightly decreasedaffinities, compared to their DOTAGA analogues (5 and 7, respectively).Between the ^(nat)Ga-complexed agents and the respective uncomplexedcompound no significant difference was observed regarding PSMAaffinities (Table 3).

TABLE 3 Binding affinities (IC₅₀ in nM) of the PSMA-SIFA ligands toPSMA. Affinities were determined using LNCaP cells (150000 cells/well)and ([¹²⁵I]I-BA)KuE (c = 0.2 nM) as the radioligand (1 h, 4° C., HBSS +1% BSA). Data are expressed as mean ± SD (n = 3, in 3 differentexperiments). IC₅₀ [nM] IC₅₀ [nM] IC₅₀ [nM] ligand ^(nat)Ga-^(nat)F-L^(nat)F-L ^(nat)Lu-^(nat)F-L 5 7.3 ± 0.2 6.4 ± 0.2 6 10.8 ± 2.5  8.5 ±1.7 7 3.0 ± 0.7 3.5 ± 0.2 3.9 ± 0.5 8 3.8 ± 0.7 2.5 ± 0.2 3.0 ± 0.2 94.5 ± 0.3 4.3 ± 0.2 10 2.8 ± 0.7 11 4.8 ± 0.7 references: (I-BA)KuE IC₅₀= 7.1 ± 2.4 nM DCFPyL IC₅₀ = 12.3 ± 1.2 nM PSMA-1007 IC₅₀ = 4.2 ± 0.5 nMInternalization

In analogy to the binding affinities, EuE-based compounds (7, 8, 9)showed significant higher internalization values compared to peptidesbearing a EuK-binding motif (5, 6). Regarding the influence of thechelator, TRAP showed to have a positive effect on internalization (5and 9, compared to 6 and 7 respectively), even though binding affinitieswhere higher for the DOTAGA analogues (Table 3). All of the ¹⁸F-labelledcompounds showed higher internalization values, compared to therespective ⁶⁸Ga-labelled tracers (Table 4).

TABLE 4 Summary of the internalized activity (c = 0.5 nM) at 1 hour as %of the reference ligand ([¹²⁵I]I-BA)KuE (c = 0.2 nM), determined onLNCaP cells (37° C., DMEM F12 + 5% BSA, 125000 cells/well). Data iscorrected for non-specific binding (10 μmol PMPA) and expressed as mean± SD (n = 3). Internalization [%] Internalization [%] Internalization[%] ligand ⁶⁸Ga-^(nat)F-L ¹⁸F-L ¹⁷⁷Lu-^(nat)F-L 5 33.0 ± 2.1 n.d. 6 43.0± 3.4 n.d. 7 126.0 ± 13.1 164.8 ± 4.5 185.8 ± 4.5 8  98.2 ± 12.4 130.3 ±5.5 165.9 ± 8.5 9 176.9 ± 11.7 211.6 ± 5.3 10 184.1 ± 16  11  134.5 ±18.5 references [%]: DCFPyL 118 ± 5 PSMA-1007 118 ± 4Human Serum Albumin Binding

TABLE 5 HSA binding of the synthesized PSMA-SIFA ligands, determined ona Chiralpak HSA column (50 × 3 mm, 5 μm, H13H-2433). HSA binding [%] HSAbinding [%] ligand ^(nat)Ga-^(nat)F-L ^(nat)Lu-^(nat)F-L 7 95.7 97.7 896.5 97.7 9 95.1 10 94.0 11 92.4Small Animal PET Imaging and Biodistribution1. [⁶⁸Ga][^(nat)F]PSMA-SIFA1 (⁶⁸Ga—^(nat)F-5)

-   -   See FIG. 2.        2. [⁶⁸Ga][^(nat)F]PSMA-SIFA2 (⁶⁸Ga—^(nat)F-6)    -   See FIG. 3.        3. PSMA-SIFA3 (7)    -   a) static ⁶⁸Ga-PET imaging    -   See FIG. 4.    -   b) dynamic ⁶⁸Ga-PET imaging    -   See FIG. 5.    -   c) static ¹⁸F-PET imaging    -   See FIG. 6.    -   d) dynamic ¹⁸F-PET imaging    -   See FIG. 7.    -   e) biodistribution studies    -   See FIG. 8.        4. PSMA-SIFA4 (8)    -   a) static ⁶⁸Ga-PET imaging    -   See FIG. 9.    -   b) dynamic ⁶⁸Ga-PET imaging    -   See FIG. 10.    -   c) static ¹⁸F-PET imaging    -   See FIG. 11.    -   d) dynamic ¹⁸F-PET imaging    -   See FIG. 12.    -   e) biodistribution studies    -   See FIG. 13.        5. PSMA-SIFA5 (9)    -   a) static ⁶⁸Ga-PET imaging    -   See FIG. 14.    -   b) dynamic ⁶⁸Ga-PET imaging    -   See FIG. 15.    -   c) static ¹⁸F-PET imaging    -   See FIG. 16.    -   d) static ¹⁸F-PET imaging    -   See FIG. 17.    -   e) biodistribution studies    -   See FIG. 18.        6. Proof of Concept Studies of ^(nat)Ga-PSMA-SIFA3 (^(nat)Ga—7)    -   a) static ¹⁸F-PET imaging    -   See FIG. 19.    -   b) biodistribution studies    -   See FIG. 20.        7. Small Animal PET Imaging Using Luthenium rhPSMA Ligands.    -   a) Static PET imaging: ¹⁸F-^(nat)Lu-rh-7    -   See FIG. 29.    -   b) Dynamic PET imaging: ¹⁸F-^(nat)Lu-rh7    -   See FIG. 30.    -   c) biodistribution studies of ¹⁷⁷Lu-^(nat)F-7, ¹⁷⁷Lu-^(nat)F-8        and ¹⁷⁷Lu-^(nat)F-10 at 24 h    -   See FIG. 31.    -   d) biodistribution of ¹⁷⁷Lu-^(nat)F-10 at 1 h and 24 h.    -   See FIG. 32.    -   e) Comparative biodistribution of established and new        rhPSMA-ligands at 24 h.    -   See FIG. 33.    -   f) Comparative biodistribution of ¹⁷⁷Lu-^(nat)F-rhPSMA-10 and        ⁶⁸Ga—^(nat)F-rhPSMA-10 at 1 h.    -   See FIG. 34.        Human PSMA-SIFA3 (7) Biodistribution and Uptake in Tumor Lesions

No adverse events or clinically detectable pharmacological effects werenoted.

FIG. 21 demonstrates the maximum intensity projection (MIR) from PET ofa subject with normal biodistribution (no tumor lesions detectable).Images were acquired 76 min post injection of 272 MBq 18F-labelledPSMA-SIFA3 (7). FIG. 21 right demonstrates the maximum intensityprojection (MIP) from PET of a subject with moderately advanced diseaseexhibiting multiple tumor lesions with high lesion-to-background ratio.Images were acquired 102 min post injection of 312 MBq 18F-labelledPSMA-SIFA3 (7).

Uptake parameters reflect background PSMA-expression for differenttissue types. Significant radiotracer uptake was only discerned forsalivary glands, kidneys, liver, spleen and duodenum. Uptake inbackground tissue was low. Uptake in tumor lesion was substantiallyhigher than in low PSMA-expressing tissue.

TABLE 6 Average SUVmax (left) and SUVmean (right) in different tissues(tissues/organs: n = 53, tumor lesions: n = 72) with its standard error.See FIG. 22 for graphical representation. SUVmax SUVmean mean min maxmean min max background 1.0 0.6 1.9 0.6 0.4 1.2 bloodpool 2.4 1.6 3.92.0 1.1 17.0 parotid gland 23.5 8.2 42.3 16.8 5.5 32.7 submandibular26.7 10.1 43.8 19.6 7.0 29.7 gland lungs 1.0 0.5 3.1 0.7 0.3 2.0 liver9.5 4.5 25.2 7.0 3.2 17.7 spleen 11.8 4.7 21 9.1 3.4 17.1 pancreas 3.91.8 9.2 2.7 1.3 7.4 duodenum 14.2 2.8 32.7 10.5 1.9 23.9 bone 1.7 0.83.1 1.1 0.6 2.1 kidney 44.3 19.1 75.2 32.1 13.2 54.7 bladder 8.3 0.5112.0 6.1 0.3 85.7 tumor lesions 26.6 4.0 95.4 19.2 2.7 71.7

Due to low background activity ratio SUV to background of organs andtumor lesions is favorable for clinical imaging. Tumor lesions aredisplayed with high contrast compared to background.

TABLE 7 Average ratio SUVmax (left) and SUVmean (right) with itsstandard error in different tissues (tissues/organs: n = 53, tumorlesions: n = 72) with its standard error. See FIG. 23 for graphicalrepresentation. ratio SUVmax ratio SUVmean to background to backgroundmean min max mean min max bloodpool 2.5 1.3 4.8 3.1 1.5 21.3 parotidgland 24.3 8.2 45.3 27.5 9.2 54.5 submandibular 27.8 10.1 54.7 32.5 11.761.8 gland lungs 1.1 0.4 3.3 1.1 0.4 4.0 liver 10.1 2.9 42.0 11.7 3.744.3 spleen 12.3 4.7 35.0 14.9 5.7 39.5 pancreas 4.0 1.5 11.3 4.3 1.910.8 duodenum 14.8 2.8 31.3 17.3 3.2 35.3 bone 1.7 0.9 2.9 1.8 1.0 3.2kidney 46.7 16.9 98.7 53.8 19.8 109.3 bladder 8.3 0.6 112.0 9.8 0.5142.8 tumor lesions 28.6 5.0 83.4 31.9 5.4 83.2

Uptake in tumor lesions and contrast to background was relatively equalbetween different types of tumors (local tumor [n=24], lymph nodemetastases [n=23], bone metastases [n=21], visceral metastases [n=4]).

TABLE 8 Average SUVmax, SUVmean, ratio SUVmax to background and ratioSUVmean to background in different tumor types with its standard error.See FIG. 24 for a graphical representation. ratio ratio SUVmax SUVmaxSUVmean SUVmean local tumor mean 26.9 29.6 19.3 32.4 min 4 5 2.7 5.4 max75.1 83.4 19.3 83.2 lymph node metastases mean 22.2 23.7 16.6 27.3 min8.1 5.8 6.4 7.1 max 67.5 63.6 44.6 70.7 bone metastses mean 31.2 32.822.2 36.6 min 7.9 7.9 5.3 8.8 max 95.4 73.4 71.7 80 visceral metastasesmean 26.1 28.9 17.4 30.2 min 20.4 18.5 15.5 22.1 max 32.5 40.6 19.1 38.2Human PSMA-SIFA3 (7) Tracer Retention in Urinary Bladder

Tracer retention in excretory urinary system is a common drawback ofPSMA-ligand imaging. 18F-labelled PSMA-SIFA3 (7) as potential leadcompound of SiFA substituted chelator-based PET agent is excreted viathe urinary excretory system, but to a much lower extent than most otherPET-agents. In addition, its retention in the bladder can besignificantly influenced by the application of furosemide at the time oftracer injection. T-test 8 revealed a statistical significantly lowertracer retention when furosemide was applied (p=0.018 both for SUVmaxand SUVmean).

TABLE 9 Average SUVmax (left) and SUVmean (right) of tracer retention inbladder in subjects with and without administration of furosemide withits standard error. See FIG. 25 for graphical representation. SUVmaxSUVmean mean min max mean min max with furosemide  4.8 1.6  32  3.4 1.126.6 without furosemide 13.9 0.5 112 10.5 0.3 85.7Clinical Results for PSMA-SIFA3 (7) Detection of Tumor Lesions andHistopathological Validation

Subjects were imaged for primary staging (n=6) and recurrent disease(n=47). Lesions indicative for prostate cancer were detected in 39patients. 72 lesions were analysed. 21 of 72 lesions had no correlate onmorphological imaging. 14 of 72 lesions measured exhibited a size ofequal or less than 5 mm on morphological imaging. Both demonstrate highclinical value of 18F-labelled PSMA-SIFA3 (7) for detection of lesionsotherwise occult on morphological imaging. Uptake parameters of the 35lesions with no correlate or small size on morphological imagingexhibited favorable uptake parameters.

TABLE 10 Average SUVmax, SUVmean, ratio SUVmax to background and ratioSUVmean to background in prostate cancer tumors with its standard error.ratio ratio SUVmax SUVmax SUVmean SUVmean mean 16.7 18.3 13.3 22.9 min9.2 5.8 6.4 7.1 max 25 43.8 28.3 56.6

Imaging examples show favorable characteristics. Both smallsubcentimeter lesions and diffuse metastatic disease involving differenttissue types are shown.

FIG. 26 shows: MIR (A) and transaxial images (B-D) of a 70 year oldpatient with biochemical recurrence 1.5 years after radicalprostatectomy (Gleason 8, pT2c, pN1). A single prostate cancer typicallesion with 5 mm diameter in right pelvis with high uptake of18F-labelled PSMA-SIFA3 (7) is present. Malignant nature of the lesionwas verified by histopathology.

FIG. 27 shows: Set of images of an 80 year old patient with progressiveadvanced castration resistant prostate cancer (PSA 66.4 ng/ml). Imagesshows high uptake of 18F-labelled PSMA-SIFA3 (7) in different classes ofprostate cancer lesions (local tumor, lymph node metastases, bonemetastases, liver metastases). Lesions demonstrated are as small as 2 mm(arrows indicate representative, not all tumor lesions).

Clinical Application of ⁶⁸Ga-Labelled PSMA-SIFA3 (7)

As a proof on concept investigation of a ⁶⁸Ga-labelled SiFA substitutedchelator-based PET tracers one subject with biochemical recurrence afterradical prostatectomy (PSA 0.44 ng/ml, pT2c, pNO, Gleason 7b) underwentPET/MR 66 min after injection of 144 MBq ⁶⁸Ga-labelled PSMA-SIFA3 (7).Uptake typical for recurrent prostate cancer is demonstrated in a 2 mmlymph node.

FIG. 28 shows proof of concept investigation of a ⁶⁸Ga-labelled SiFAsubstituted chelator-based PET tracer.

Human PSMA-SIFA3 (7) Studies

1. Biodistribution and Uptake in Tumor Lesions

-   -   (a) Maximum intensity projection from PET of a subject with        normal biodistribution.    -   See FIG. 21.    -   (b) Average standardized uptake values in different tissues    -   See FIG. 22.    -   (c) Average ratio standardized uptake values in different        tissues    -   See FIG. 23.    -   (d) Average standardized uptake values in different tumor types    -   See FIG. 24.        2. Tracer Retention in Urinary Bladder    -   (a) Average standardized uptake values of tracer retention in        bladder    -   See FIG. 25.        3. Clinical Results for Detection of Tumor Lesions and        Histopathological Validation    -   See FIGS. 26 and 27.        4. Clinical Application of ⁶⁸Ga-Labelled PSMA-SIFA3 (7)    -   See FIG. 28.

The invention claimed is:
 1. A compound selected from the groupconsisting of:

or a pharmaceutically acceptable salt and or individual isomer thereof,optionally containing a chelated nonradioactive or radioactive cationand wherein the fluorine atom is optionally ¹⁸F.
 2. A pharmaceutical ordiagnostic composition comprising or consisting of one or more compoundsaccording to claim 1, or a pharmaceutically acceptable salt orindividual isomer thereof.
 3. A method of imaging and/or diagnosingcancer comprising administering a compound according to claim 1, or apharmaceutically acceptable salt or individual isomer thereof, to apatient in need thereof; and imaging the patient in need thereof.
 4. Amethod of treating cancer in a patient, comprising administering aneffective amount of a compound according to claim 1, or apharmaceutically acceptable salt or individual isomer thereof, to apatient.
 5. A method of reducing neoangiogenesis or angiogenesis in apatient in need thereof, comprising administering an effective amount ofa compound according to claim 1, or a pharmaceutically acceptable saltor individual isomer thereof, to a patient in need thereof.
 6. Themethod according to claim 4, wherein the cancer is prostate, breast,lung, colorectal or renal cell carcinoma.
 7. A method of imaging and/ordiagnosing neoangiogenesis or angiogenesis in a patient, comprisingadministering a compound according to claim 1, or a pharmaceuticallyacceptable salt or individual isomer thereof, to a patient in needthereof; and imaging the patient in need thereof.
 8. The compound ofclaim 1, wherein the compound is

or a pharmaceutically acceptable salt or individual isomer thereof,optionally containing a chelated nonradioactive or radioactive cationand wherein the fluorine atom is optionally ¹⁸F.
 9. The compound ofclaim 1, wherein the compound is

or a pharmaceutically acceptable salt or individual isomer thereof,optionally containing a chelated nonradioactive or radioactive cationand wherein the fluorine atom is optionally ¹⁸F.
 10. The compound ofclaim 1, wherein the compound is

or a pharmaceutically acceptable salt or individual isomer thereof,optionally containing a chelated nonradioactive or radioactive cationand wherein the fluorine atom is optionally ¹⁸F.
 11. The compound ofclaim 1, wherein the compound is

or a pharmaceutically acceptable salt or individual isomer thereof,optionally containing a chelated nonradioactive or radioactive cationand wherein the fluorine atom is optionally ¹⁸F.
 12. The compound ofclaim 1, wherein the compound is

or a pharmaceutically acceptable salt or individual isomer thereof,optionally containing a chelated nonradioactive or radioactive cationand wherein the fluorine atom is optionally ¹⁸F.
 13. The compound ofclaim 1, wherein the compound is

or a pharmaceutically acceptable salt or individual isomer thereof,optionally containing a chelated nonradioactive or radioactive cationand wherein the fluorine atom is optionally ¹⁸F.
 14. The compound ofclaim 1, wherein the compound is

or a pharmaceutically acceptable salt or individual isomer thereof,optionally containing a chelated nonradioactive or radioactive cationand wherein the fluorine atom is optionally ¹⁸F.
 15. The compoundaccording to claim 1, or a pharmaceutically acceptable salt orindividual isomer thereof, wherein the chelated cation is selected fromthe cations of ⁴³Sc, ⁴⁴SC, ⁴⁷Sc, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁹⁰Y, ¹¹¹In,¹⁴⁹Tb, ¹⁵²Tb, ¹⁵⁵Tb, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ²¹²Pb, ²¹²Bi,²¹³Bi, ²²⁵Ac, and ²²⁷Th or a cationic molecule comprising ¹⁸F.
 16. Thecompound according to claim 1, or a pharmaceutically acceptable salt orindividual isomer thereof, wherein the fluorine atom is ¹⁸F.